Amino acid sequences directed against the angiopoietin/tie system and polypeptides comprising the same for the treatment of diseases and disorders related to angiogenesis

ABSTRACT

The present invention relates to amino acid sequences that are directed against proteins from the group of the Angiopoietin/Tie family such as Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl12, Angptl13, Angptl14, Angptl15, Angptl16, as well as to compounds or constructs, and in particular proteins and polypeptides, that comprise or essentially consist of one or more of such amino acid sequences.

The present invention relates to amino acid sequences that are directedagainst proteins from the group of the Angiopoietin/Tie family such asTie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4,Angptl5, Angptl6, as well as to compounds or constructs, and inparticular proteins and polypeptides, that comprise or essentiallyconsist of one or more of such amino acid sequences.

The invention also relates to nucleic acids encoding such amino acidsequences and polypeptides (also referred to herein as “nucleic acids ofthe invention” or “nucleotide sequences of the invention”); to methodsfor preparing such amino acid sequences and polypeptides; to host cellsexpressing or capable of expressing such amino acid sequences orpolypeptides; to compositions, and in particular to pharmaceuticalcompositions, that comprise such amino acid sequences, polypeptides,nucleic acids and/or host cells; and to uses of such amino acidsequences or polypeptides, nucleic acids, host cells and/orcompositions, in particular for prophylactic, therapeutic or diagnosticpurposes, such as the prophylactic, therapeutic or diagnostic purposesmentioned herein.

Other aspects, embodiments, advantages and applications of the inventionwill become clear from the further description herein.

Angiopoietins 1-4 (Ang1-Ang4) constitute a family of growth factors thatfunction as ligands of Tie2, a Receptor Tyrosine Kinase (RTK) expressedmainly in endothelial cells. Angs/Tie2 signaling is involved in multiplesteps of angiogenesis such as the destabilization of existing vesselsand endothelial cell migration (Bouis et al 2006). Ang1 and 4 have beenshown to act as obligatory agonists promoting structural integrity ofblood vessels, whereas Ang2 and Ang3 function as a context-dependentantagonist or agonist. In spite of the structural homology with Tie2,none of the known Angs bind to another RTK named Tie1, although somestudies indicate an essential role for Tie1 in vascular development(Eklund L., Olsen B. R. Tie receptors and their angiopoietin ligands arecontext-dependent regulators of vascular remodeling. Experimental. CellResearch (2006) 312: 630-641). Based in their similarity in structurewith Angs six angiopoietin-like proteins (Angptls) have beingidentified. Interestingly, Angptls also function in angiogenesis throughregulating survival and migration of endothelial cells, although theseproteins do not bind the angiopoietin receptor Tie2 (Oike Y, Akao M.,Kubota E, Suda T. Angiopoietin-like proteins: potential new targets formetabolic syndrome therapy. TRENDS in Molecular Medicine (2005). 11:473-479; Bouis D, Kusumanto Y, Meijer C, Mulder N H, Hospers G A P. Areview on pro- and anti-angiogenic factors as targets of clinicalintervention. Pharmalogical Research 53 (2006) 89-103, Review).Deregulated angiogenesis leads to numerous malignant, ischemic,inflammatory, infectious and immune disorders (Carmeliet P. Angiogenesisin health and disease. Nature Medicine 9 (2003) 653-660) and therefore,the modulation of Tie receptors, Angs and Angptls may have manyinteresting potential therapeutic applications.

Tie receptors are endothelial-specific RTKs that share a high degree ofhomology. The extracellular regions of both receptors Tie1 and Tie2,with 33% similarity, contain an immunoglobulin-like loop, three EGF-likedomains, a second Ig-like loop, and three fibronectin type III repeats.The cytoplasmic regions of both receptors, presenting 76% of similarity,contain tyrosine kinase domains including a number of phosphorylationand protein interaction sites (Thurston G. Role of Angiopoietins and Tiereceptor tyrosine kinases in angiogenesis and lymphangiogenesis. CellTissue Res (2003) 314:61-68; Fiedler U., Augustin H. G. Angiopoietins: alink between angiogenesis and inflammation. TRENDS in Immunology. (2006)27:552-558). Signaling through dimerisation and autophosphorylation ofTie2 upon binding of agonist Angs has been studied and results suggestthat the major signalling pathway involves activation ofphosphatidylinositol 3′ kinase (Eklund and Olsen, 2006, supra). Also, aswill be clear from the further disclosure herein, and depending on theTie against which they are directed and their desired (therapeutic)effect, the amino acid sequences, Nanobodies and polypeptides of theinvention may act as (full or partial) agonists, (full or partial, andcompetitive or non-competitive) antagonists or as inverse agonists ofTie, e.g. Tie2 and/or of the biological function, pathway, mechanism,effect, signalling or response associated therewith. They may do so inan irreversible but preferably reversible manner.

Angs contain an amino-terminal angiopoietin-specific domain followed bya coiled-coil domain, a linker peptide and a carboxy-terminal fibrinogenhomology domain. The fibrinogen homology domain is responsible forreceptor binding, the coiled-coil domain is required for dimerization ofangiopoietin monomers and the short amino-terminal region formsring-like structures that cluster dimers into variable sized multimersnecessary for Tie2 activation (Eklund and Olsen, 2006, supra). HumanAng1 shares approximately 97% amino acid sequence identity with mouseAng-1, while human and mouse Ang2 share only 85% amino acid sequenceidentity. Mouse and human Ang2 are 60% identical to their Ang1 homologs.In the case of human Ang4 it shares 45%. 47% and 54% amino acid sequenceidentity with human Ang1, human Ang2 and mouse Ang3 respectively.Structurally very similar to Angs, Angptls contain a coiled-coileddomain and a fibrinogen-like domain similar to those found in Angs.

List of Tie, Angs and Angptls: (Bouis et al., 2006; Eklund and Olsen,2006; Oike et al., 2005, supra)

Tie-family Tie1 Tie2 Angs-family Ang1 Ang2 Ang3 Ang4 Angptls-familyAngptl1 Angptl2 Angptl3 Angptl4 Angptl5 Angptl6

Angiogenesis plays a major role in several pathologic processes astumour vascularisation, diabetic retinopathy, psoriasis and reumathoidarthritis, where pro- and anti-angiogenic angiopoietins and Tiereceptors are widely expressed (Bach F., Uddin Burke D. Angiopoietins inmalignancy. EJSO (2007). 33:7-15; Pandya N. M., Dhalla N. S., Santani D.D. Angiogenesis—a new target for future therapy. Vascular Pharmacology(2006) 44: 265-274; Carmeliet, 2003, supra). Many anti-angiogenicfactors targeting Tie and angiopoietins are in development. It has beenreported that modulation of the expression and inhibition of theseangiogenesis-related proteins caused a reduction on tumour growth andmetastasis by inhibiting tumour angiogenesis (Bach et al., 2007, supra;Onliner J. et al., Suppression of angiogenesis and tumor growth byselective inhibition of angiopoietin-2. Cancer Cell (2004), 6: 507-516;Bouis et al. 2006, supra). The role of Ang2 is not that clear since itis context dependent. It seems that in a non-pathological situationfunction Ang2 works as an antagonist and the ratio Ang1:Ang2 is 1:1 butin malignancy with tumor angiogenesis the expression of Ang2 increases.

Furthermore, a pro-angiogenic therapy could be beneficial in treatmentof ischemic diseases.

The polypeptides and/or compositions of the present invention cangenerally be used to modulate, and in particular inhibit and/or preventof the angiopoietin-Tie interactions and in particular the binding ofangiopoietin ligands (Ang 1 to 4) to receptor Tie1 and/or Tie2, and thusto modulate, and in particular inhibit or prevent, the signalling thatis mediated by said interactions, to modulate the biological pathways inwhich ligands and/or targets are involved, and/or to modulate thebiological mechanisms, responses and effects associated with suchsignalling or these pathways. Similarly, angiopoietin-like ligands(Angptl to Angptl6) interactions may be disrupted by polypeptides and/orcompositions of the present invention.

As such, the polypeptides and compositions of the present invention canbe used for the prevention and treatment of diseases and disordersrelated to angiogenesis. Generally, “said diseases and disorders relatedto angiogensis” can be defined as diseases and disorders that can beprevented and/or treated, respectively, by suitably administering to asubject in need thereof (i.e. having the disease or disorder or at leastone symptom thereof and/or at risk of attracting or developing thedisease or disorder) of either a polypeptide or composition of theinvention (and in particular, of a pharmaceutically active amountthereof) and/or of a known active principle active against theangiopoietin/Tie system or a biological pathway or mechanism in whichsaid system is involved (and in particular, of a pharmaceutically activeamount thereof). Examples of such diseases and disorders will be clearto the skilled person based on the disclosure herein, and for exampleinclude the following diseases and disorders:

Cancer and Angiopoietins:

-   Bach F, Uddin F J., Burke D. Angiopoietins in malignancy. EJSO    (2007). 33:7-15.

Cancer and Tie Receptors:

-   Blume-Jensen P and Hunter T. Oncogenic kinase signaling. Nature    (2001). 44: 355-365.

Diabetic Retinopathy and Angiopoietins:

-   Patel J. I., Hykin P. G., Gregor Z. J., Boulton M. and Cree I. A.    Angiopoietin concentrations in diabetic retinopathy. Br. J.    Ophthalmol (2005). 89: 480-483.

Rheumatoid Arthritis and Tie2 and Angiopoietins:

-   DeBusk L. M., Chen Y., Nishishita T., Chen J., Thomas J. W.,    Lin P. C. Tie2 receptor tyrosine kinase, a major mediator of tumor    necrosis factor a-induced angiogenesis in rheumatoid arthritis.    ARTHRITIS& RHEUMATISM (2003). 48: 2461-2471.-   Shahrara S., Volin M. V., Connors M. A., Haines G. K., Koch A. E.    Differential expression of the angiogenic Tie receptor family in    arthritic and normal synovial tissue. Arthritis Res (2002) 4:    201-208.

Psoriasis and Tie2 and Angiopoietins:

-   Kuroda K., Sapadin A., Shoji T., Fleischmajer R., Lebwohl M. Altered    expression of angiopoietins and Tie2 endothelium receptor in    psoriasis. The journal of investigate dermatology. (2001). 116:    713-720.

Ischemia, Renal Carcinoma and Angptl4:

-   LeJan S., Amy C., Cazes A., Monnot C., Lamande N., Favier J.,    Philippe J., Sibony M., Gasc 1-M, Corvol P., Germain S.    Angiopoietin-like 4 is a proangiogenic factor produced during    Ischemia and conventional renal cell carcinoma. American Journal of    Pathology (2003) 162: 1521-1528.

In particular, the polypeptides and compositions of the presentinvention can be used for the prevention and/or treatment of diseasesand disorders related to angiogenesis which are characterized byexcessive and/or unwanted creation of blood vessels or lack of creationof blood vessels. Examples of such disorders are cardiovasculardisorders, cancers, diabetic retinopathy, wound healing, rheumatoidarthritis, obesity, alveolarization and psoriasis.

Thus, without being limited thereto, the amino acid sequences andpolypeptides of the invention can for example be used to prevent and/orto treat all diseases and disorders that are currently being preventedor treated with active principles that can modulate angiogenesis, suchas those mentioned in the prior art cited above and others. It is alsoenvisaged that the polypeptides of the invention can be used to preventand/or to treat all diseases and disorders for which treatment with suchactive principles is currently being developed, has been proposed, orwill be proposed or developed in future. In addition, it is envisagedthat, because of their favourable properties as further describedherein, the polypeptides of the present invention may be used for theprevention and treatment of other diseases and disorders than those forwhich these known active principles are being used or will be proposedor developed; and/or that the polypeptides of the present invention mayprovide new methods and regimens for treating the diseases and disordersdescribed herein.

Other applications and uses of the amino acid sequences and polypeptidesof the invention will become clear to the skilled person from thefurther disclosure herein.

Generally, it is an object of the invention to provide pharmacologicallyactive agents, as well as compositions comprising the same, that can beused in the diagnosis, prevention and/or treatment of cancers, diabeticretinopathy, wound healing, rheumatoid arthritis, obesity,alveolarization and psoriasis and of the further diseases and disordersmentioned herein; and to provide methods for the diagnosis, preventionand/or treatment of such diseases and disorders that involve theadministration and/or use of such agents and compositions.

In particular, it is an object of the invention to provide suchpharmacologically active agents, compositions and/or methods that havecertain advantages compared to the agents, compositions and/or methodsthat are currently used and/or known in the art. These advantages willbecome clear from the further description below.

More in particular, it is an object of the invention to providetherapeutic proteins that can be used as pharmacologically activeagents, as well as compositions comprising the same, for the diagnosis,prevention and/or treatment cancers, diabetic retinopathy, woundhealing, rheumatoid arthritis, obesity, alveolarization and psoriasisand of the further diseases and disorders mentioned herein; and toprovide methods for the diagnosis, prevention and/or treatment of suchdiseases and disorders that involve the administration and/or the use ofsuch therapeutic proteins and compositions.

Accordingly, it is a specific object of the present invention to provideamino acid sequences that are directed against Tie1, Tie2, Ang1, Ang2,Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6 (asdefined herein), in particular against Tie2, Ang1, Ang2, Ang4 or Angptl4from a warm-blooded animal, more in particular Tie2, Ang1, Ang2, Ang4 orAngptl4 from a mammal, and especially against human Tie2, Ang1, Ang2,Ang4 or Angptl4; and to provide proteins and polypeptides comprising oressentially consisting of at least one such amino acid sequence.

In particular, it is a specific object of the present invention toprovide such amino acid sequences and such proteins and/or polypeptidesthat are suitable for prophylactic, therapeutic and/or diagnostic use ina warm-blooded animal, and in particular in a mammal, and more inparticular in a human being.

More in particular, it is a specific object of the present invention toprovide such amino acid sequences and such proteins and/or polypeptidesthat can be used for the prevention, treatment, alleviation and/ordiagnosis of one or more diseases, disorders or conditions associatedwith Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3,Angptl4, Angptl5, Angptl6 and/or mediated by Tie1, Tie2, Ang1, Ang2,Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6(such as the diseases, disorders and conditions mentioned herein) in awarm-blooded animal, in particular in a mammal, and more in particularin a human being.

It is also a specific object of the invention to provide such amino acidsequences and such proteins and/or polypeptides that can be used in thepreparation of pharmaceutical or veterinary compositions for theprevention and/or treatment of one or more diseases, disorders orconditions associated with and/or mediated by Tie1, Tie2, Ang1, Ang2,Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6(such as the diseases, disorders and conditions mentioned herein) in awarm-blooded animal, in particular in a mammal, and more in particularin a human being.

In the invention, generally, these objects are achieved by the use ofthe amino acid sequences, proteins, polypeptides and compositions thatare described herein.

In general, the invention provides amino acid sequences that aredirected against (as defined herein) and/or can specifically bind (asdefined herein) to Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,Angptl3, Angptl4, Angptl5, or Angptl6; as well as compounds andconstructs, and in particular proteins and polypeptides, that compriseat least one such amino acid sequence.

More in particular, the invention provides amino acid sequences that canbind to Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3,Angptl4, Angptl5, or Angptl6 with an affinity (suitably measured and/orexpressed as a K_(D)-value (actual or apparent), a K_(A)-value (actualor apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively asan IC₅₀ value, as further described herein) that is as defined herein;as well as compounds and constructs, and in particular proteins andpolypeptides, that comprise at least one such amino acid sequence.

In particular, amino acid sequences and polypeptides of the inventionare preferably such that they:

-   -   bind to Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,        Angptl3, Angptl4, Angptl5, or Angptl6 with a dissociation        constant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or less, and        preferably 10⁻⁷ to 10⁻¹² moles/liter or less and more preferably        10⁻⁸ to 10⁻¹² moles/liter (i.e. with an association constant        (K_(A)) of 10⁵ to 10¹² liter/moles or more, and preferably 10⁷        to 10¹² liter/moles or more and more preferably 10⁸ to 10¹²        liter/moles);        and/or such that they:    -   bind to Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,        Angptl3, Angptl4, Angptl5, or Angptl6 with a k_(on)-rate of        between 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, preferably between 10³        M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more preferably between 10⁴ M⁻¹s⁻¹ and        10⁷ M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹;        and/or such that they:    -   bind to Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,        Angptl3, Angptl4, Angptl5, or Angptl6 with a k_(off) rate        between 1 s⁻¹ (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near        irreversible complex with a t_(1/2) of multiple days),        preferably between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably        between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as between 10⁻⁴ s⁻¹ and 10⁻⁶        s⁻¹.

Preferably, a monovalent amino acid sequence of the invention (or apolypeptide that contains only one amino acid sequence of the invention)is preferably such that it will bind to Tie1, Tie2, Ang1, Ang2, Ang3,Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6 with anaffinity less than 500 nM, preferably less than 200 nM, more preferablyless than 10 nM, such as less than 500 μM.

Some preferred IC50 values for binding of the amino acid sequences orpolypeptides of the invention to Tie1, Tie2, Ang1, Ang2, Ang3, Ang4,Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6 will becomeclear from the further description and examples herein.

For binding to Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,Angptl3, Angptl4, Angptl5, or Angptl6, an amino acid sequence of theinvention will usually contain within its amino acid sequence one ormore amino acid residues or one or more stretches of amino acid residues(i.e. with each “stretch” comprising two or amino acid residues that areadjacent to each other or in close proximity to each other, i.e. in theprimary or tertiary structure of the amino acid sequence) via which theamino acid sequence of the invention can bind to Tie1, Tie2, Ang1, Ang2,Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6,which amino acid residues or stretches of amino acid residues thus formthe “site” for binding to Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1,Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6 (also referred to hereinas the “antigen binding site”).

The amino acid sequences provided by the invention are preferably inessentially isolated form (as defined herein), or form part of a proteinor polypeptide of the invention (as defined herein), which may compriseor essentially consist of one or more amino acid sequences of theinvention and which may optionally further comprise one or more furtheramino acid sequences (all optionally linked via one or more suitablelinkers). For example, and without limitation, the one or more aminoacid sequences of the invention may be used as a binding unit in such aprotein or polypeptide, which may optionally contain one or more furtheramino acid sequences that can serve as a binding unit (i.e. against oneor more other targets than Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1,Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, so as to provide amonovalent, multivalent or multispecific polypeptide of the invention,respectively, all as described herein. Such a protein or polypeptide mayalso be in essentially isolated form (as defined herein).

The amino acid sequences and polypeptides of the invention as suchpreferably essentially consist of a single amino acid chain that is notlinked via disulphide bridges to any other amino acid sequence or chain(but that may or may not contain one or more intramolecular disulphidebridges. For example, it is known that Nanobodies—as describedherein—may sometimes contain a disulphide bridge between CDR3 and CDR1or FR2). However, it should be noted that one or more amino acidsequences of the invention may be linked to each other and/or to otheramino acid sequences (e.g. via disulphide bridges) to provide peptideconstructs that may also be useful in the invention (for example Fab′fragments, F(ab′)₂ fragments, ScFv constructs, “diabodies” and othermultispecific constructs. Reference is for example made to the review byHolliger and Hudson, Nat. Biotechnol. 2005 September; 23(9):1126-36).

Generally, when an amino acid sequence of the invention (or a compound,construct or polypeptide comprising the same) is intended foradministration to a subject (for example for therapeutic and/ordiagnostic purposes as described herein), it is preferably either anamino acid sequence that does not occur naturally in said subject; or,when it does occur naturally in said subject, in essentially isolatedform (as defined herein).

It will also be clear to the skilled person that for pharmaceutical use,the amino acid sequences of the invention (as well as compounds,constructs and polypeptides comprising the same) are preferably directedagainst human Tie2, Ang1, Ang2, or Ang4; whereas for veterinarypurposes, the amino acid sequences and polypeptides of the invention arepreferably directed against Tie2, Ang1, Ang2, Ang4 or Angptl4 from thespecies to be treated, or at least cross-reactive with Tie2, Ang1, Ang2,Ang4 or Angptl4 from the species to be treated.

Furthermore, an amino acid sequence of the invention may optionally, andin addition to the at least one binding site for binding against Tie1,Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4,Angptl5, or Angptl6, contain one or more further binding sites forbinding against other antigens, proteins or targets.

The efficacy of the amino acid sequences and polypeptides of theinvention, and of compositions comprising the same, can be tested usingany suitable in vitro assay, cell-based assay, in vivo assay and/oranimal model known per se, or any combination thereof, depending on thespecific disease or disorder involved. Suitable assays and animal modelswill be clear to the skilled person, and for example include Solid-phasereceptor binding and blocking assays, Receptor activation/inactivationassays, In vivo angiogenesis assay, In vivo direct anti angiogeniceffect, Lipoprotein lipase (LPL) assay, In vivo .CAM (chickchorioallantoric membrane) assay, In vivo animal model studies as wellas the assays and animal models used in the experimental part below andin the prior art cited herein.

Also, according to the invention, amino acid sequences and polypeptidesthat are directed against Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1,Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6 from a first species ofwarm-blooded animal may or may not show cross-reactivity with Tie1,Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4,Angptl5, or Angptl6 from one or more other species of warm-bloodedanimal. For example, amino acid sequences and polypeptides directedagainst human Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,Angptl3, Angptl4, Angptl5, or Angptl6 may or may not show crossreactivity with Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,Angptl3, Angptl4, Angptl5, or Angptl6 from one or more other species ofprimates (such as, without limitation, monkeys from the genus Macaca(such as, and in particular, cynomologus monkeys (Macaca fascicularis)and/or rhesus monkeys (Macaca mulatta)) and baboon (Papio ursinus))and/or with Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,Angptl3, Angptl4, Angptl5, or Angptl6 from one or more species ofanimals that are often used in animal models for diseases (for examplemouse, rat, rabbit, pig or dog), and in particular in animal models fordiseases and disorders associated with Tie1, Tie2, Ang1, Ang2, Ang3,Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6 (such asthe species and animal models mentioned herein). In this respect, itwill be clear to the skilled person that such cross-reactivity, whenpresent, may have advantages from a drug development point of view,since it allows the amino acid sequences and polypeptides against humanTie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl1, Angptl3, Angptl4,Angptl5, or Angptl6 to be tested in such disease models.

More generally, amino acid sequences and polypeptides of the inventionthat are cross-reactive with Tie1, Tie2, Ang1, Ang2, Ang3, Ang4,Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6 from multiplespecies of mammal will usually be advantageous for use in veterinaryapplications, since it will allow the same amino acid sequence orpolypeptide to be used across multiple species. Thus, it is alsoencompassed within the scope of the invention that amino acid sequencesand polypeptides directed against Tie1, Tie2, Ang1, Ang2, Ang3, Ang4,Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6 from one speciesof animal (such as amino acid sequences and polypeptides against humanTie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4,Angptl5, or Angptl6 can be used in the treatment of another species ofanimal, as long as the use of the amino acid sequences and/orpolypeptides provide the desired effects in the species to be treated.

The present invention is in its broadest sense also not particularlylimited to or defined by a specific antigenic determinant, epitope,part, domain, subunit or confirmation (where applicable) of Tie1, Tie2,Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6 against which the amino acid sequences and polypeptides of theinvention are directed. However, it is generally assumed and preferredthat the amino acid sequences and polypeptides of the invention arepreferably directed against the Ang1 binding site on Tie2, or the Tie2binding site on Ang2—see experimental part. Thus, in one preferred, butnon-limiting aspect, the amino acid sequences and polypeptides of theinvention are directed against Ang1 binding site of Tie2 or the Tie2binding site of Ang2, and are as further defined herein.

It is also within the scope of the invention that, where applicable, anamino acid sequence of the invention can bind to two or more antigenicdeterminants, epitopes, parts, domains, subunits or confirmations ofTie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4,Angptl5, or Angptl6. In such a case, the antigenic determinants,epitopes, parts, domains or subunits of Tie1, Tie2, Ang1, Ang2, Ang3,Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6 to whichthe amino acid sequences and/or polypeptides of the invention bind maybe essentially the same (for example, if Tie1, Tie2, Ang1, Ang2, Ang3,Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6 containsrepeated structural motifs or occurs in a multimeric form) or may bedifferent (and in the latter case, the amino acid sequences andpolypeptides of the invention may bind to such different antigenicdeterminants, epitopes, parts, domains, subunits of Tie1, Tie2, Ang1,Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6 with an affinity and/or specificity which may be the same ordifferent). Also, far example, when Tie1, Tie2, Ang1, Ang2, Ang3, Ang4,Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6 exists in anactivated conformation and in an inactive conformation, the amino acidsequences and polypeptides of the invention may bind to either one ofthese confirmation, or may bind to both these confirmations (i.e. withan affinity and/or specificity which may be the same or different).Also, for example, the amino acid sequences and polypeptides of theinvention may bind to a conformation of Tie1, Tie2, Ang1, Ang2, Ang3,Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6 in whichit is bound to a pertinent ligand, may bind to a conformation of Tie1,Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4,Angptl5, or Angptl6 in which it not bound to a pertinent ligand, or maybind to both such conformations (again with an affinity and/orspecificity which may be the same or different).

It is also expected that the amino acid sequences and polypeptides ofthe invention will generally bind to all naturally occurring orsynthetic analogs, variants, mutants, alleles, parts and fragments ofTie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4,Angptl5, or Angptl6; or at least to those analogs, variants, mutants,alleles, parts and fragments of Tie1, Tie2, Ang1, Ang2, Ang3, Ang4,Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6 that contain oneor more antigenic determinants or epitopes that are essentially the sameas the antigenic determinant(s) or epitope(s) to which the amino acidsequences and polypeptides of the invention bind in Tie1, Tie2, Ang1,Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6 (e.g. in wild-type Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1,Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6). Again, in such a case,the amino acid sequences and polypeptides of the invention may bind tosuch analogs, variants, mutants, alleles, parts and fragments with anaffinity and/or specificity that are the same as, or that are differentfrom (i.e. higher than or lower than), the affinity and specificity withwhich the amino acid sequences of the invention bind to (wild-type)Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4,Angptl5, or Angptl6. It is also included within the scope of theinvention that the amino acid sequences and polypeptides of theinvention bind to some analogs, variants, mutants, alleles, parts andfragments of Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,Angptl3, Angptl4, Angptl5, or Angptl6, but not to others.

When Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3,Angptl4, Angptl5, or Angptl6 exists in a monomeric form and in one ormore multimeric forms, it is within the scope of the invention that theamino acid sequences and polypeptides of the invention only bind toTie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4,Angptl5, or Angptl6 in monomeric form, only bind to Tie1, Tie2, Ang1,Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6 in multimeric form, or bind to both the monomeric and themultimeric form. Again, in such a case, the amino acid sequences andpolypeptides of the invention may bind to the monomeric form with anaffinity and/or specificity that are the same as, or that are differentfrom (i.e. higher than or lower than), the affinity and specificity withwhich the amino acid sequences of the invention bind to the multimericform.

Also, when Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,Angptl3, Angptl4, Angptl5, or Angptl6 can associate with other proteinsor polypeptides to form protein complexes (e.g. with multiple subunits),it is within the scope of the invention that the amino acid sequencesand polypeptides of the invention bind to Tie1, Tie2, Ang1, Ang2, Ang3,Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6 in itsnon-associated state, bind to Tie1, Tie2, Ang1, Ang2, Ang3, Ang4,Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6 in itsassociated state, or bind to both. In all these cases, the amino acidsequences and polypeptides of the invention may bind to such multimersor associated protein complexes with an affinity and/or specificity thatmay be the same as or different from (i.e. higher than or lower than)the affinity and/or specificity with which the amino acid sequences andpolypeptides of the invention bind to Tie1, Tie2, Ang1, Ang2, Ang3,Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6 in itsmonomeric and non-associated state.

Also, as will be clear to the skilled person, proteins or polypeptidesthat contain two or more amino acid sequences directed against Tie1,Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4,Angptl5, or Angptl6 may bind with higher avidity to Tie1, Tie2, Ang1,Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6 than the corresponding monomeric amino acid sequence(s). Forexample, and without limitation, proteins or polypeptides that containtwo or more amino acid sequences directed against different epitopes ofTie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4,Angptl5, or Angptl6 may (and usually will) bind with higher avidity thaneach of the different monomers, and proteins or polypeptides thatcontain two or more amino acid sequences directed against Tie1, Tie2,Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6 may (and usually will) bind also with higher avidity to amultimer of Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,Angptl3, Angptl4, Angptl5, or Angptl6.

Generally, amino acid sequences and polypeptides of the invention willat least bind to those forms of Tie1, Tie2, Ang1, Ang2, Ang3, Ang4,Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6 (includingmonomeric, multimeric and associated forms) that are the most relevantfrom a biological and/or therapeutic point of view, as will be clear tothe skilled person.

It is also within the scope of the invention to use parts, fragments,analogs, mutants, variants, alleles and/or derivatives of the amino acidsequences and polypeptides of the invention, and/or to use proteins orpolypeptides comprising or essentially consisting of one or more of suchparts, fragments, analogs, mutants, variants, alleles and/orderivatives, as long as these are suitable for the uses envisagedherein. Such parts, fragments, analogs, mutants, variants, allelesand/or derivatives will usually contain (at least part of) a functionalantigen-binding site for binding against Tie1, Tie2, Ang1, Ang2, Ang3,Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6; and morepreferably will be capable of specific binding to Tie1, Tie2, Ang1,Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6, and even more preferably capable of binding to Tie1, Tie2,Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6 with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein. Some non-limitingexamples of such parts, fragments, analogs, mutants, variants, alleles,derivatives, proteins and/or polypeptides will become clear from thefurther description herein. Additional fragments or polypeptides of theinvention may also be provided by suitably combining (i.e. by linking orgenetic fusion) one or more (smaller) parts or fragments as describedherein.

In one specific, but non-limiting aspect of the invention, which will befurther described herein, such analogs, mutants, variants, alleles,derivatives have an increased half-life in serum (as further describedherein) compared to the amino acid sequence from which they have beenderived. For example, an amino acid sequence of the invention may belinked (chemically or otherwise) to one or more groups or moieties thatextend the half-life (such as PEG), so as to provide a derivative of anamino acid sequence of the invention with increased half-life.

In one specific, but non-limiting aspect, the amino acid sequence of theinvention may be an amino acid sequence that comprises an immunoglobulinfold or may be an amino acid sequence that, under suitable conditions(such as physiological conditions) is capable of forming animmunoglobulin fold (i.e. by folding). Reference is inter alia made tothe review by Halaby et al., J. (1999) Protein Eng. 12, 563-71.Preferably, when properly folded so as to form an immunoglobulin fold,such an amino acid sequence is capable of specific binding (as definedherein) to Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,Angptl3, Angptl4, Angptl5, or Angptl6; and more preferably capable ofbinding to Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,Angptl3, Angptl4, Angptl5, or Angptl6 with an affinity (suitablymeasured and/or expressed as a K_(D)-value (actual or apparent), aK_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate,or alternatively as an IC₅₀ value, as further described herein) that isas defined herein. Also, parts, fragments, analogs, mutants, variants,alleles and/or derivatives of such amino acid sequences are preferablysuch that they comprise an immunoglobulin fold or are capable forforming, under suitable conditions, an immunoglobulin fold.

In particular, but without limitation, the amino acid sequences of theinvention may be amino acid sequences that essentially consist of 4framework regions (FR1 to FR4 respectively) and 3 complementaritydetermining regions (CDR1 to CDR3 respectively); or any suitablefragment of such an amino acid sequence (which will then usually containat least some of the amino acid residues that form at least one of theCDR's, as further described herein).

The amino acid sequences of the invention may in particular be animmunoglobulin sequence or a suitable fragment thereof, and more inparticular be an immunoglobulin variable domain sequence or a suitablefragment thereof, such as light chain variable domain sequence (e.g. aV_(L)-sequence) or a suitable fragment thereof; or a heavy chainvariable domain sequence (e.g. a V_(H)-sequence) or a suitable fragmentthereof. When the amino acid sequence of the invention is a heavy chainvariable domain sequence, it may be a heavy chain variable domainsequence that is derived from a conventional four-chain antibody (suchas, without limitation, a V_(H) sequence that is derived from a humanantibody) or be a so-called V_(HH)-sequence (as defined herein) that isderived from a so-called “heavy chain antibody” (as defined herein).

However, it should be noted that the invention is not limited as to theorigin of the amino acid sequence of the invention (or of the nucleotidesequence of the invention used to express it), nor as to the way thatthe amino acid sequence or nucleotide sequence of the invention is (orhas been) generated or obtained. Thus, the amino acid sequences of theinvention may be naturally occurring amino acid sequences (from anysuitable species) or synthetic or semi-synthetic amino acid sequences.In a specific but non-limiting aspect of the invention, the amino acidsequence is a naturally occurring immunoglobulin sequence (from anysuitable species) or a synthetic or semi-synthetic immunoglobulinsequence, including but not limited to “humanized” (as defined herein)immunoglobulin sequences (such as partially or fully humanized mouse orrabbit immunoglobulin sequences, and in particular partially or fullyhumanized. V_(HH) sequences or Nanobodies), “camelized” (as definedherein) immunoglobulin sequences, as well as immunoglobulin sequencesthat have been obtained by techniques such as affinity maturation (forexample, starting from synthetic, random or naturally occurringimmunoglobulin sequences), CDR grafting, veneering, combining fragmentsderived from different immunoglobulin sequences, PCR assembly usingoverlapping primers, and similar techniques for engineeringimmunoglobulin sequences well known to the skilled person; or anysuitable combination of any of the foregoing. Reference is for examplemade to the standard handbooks, as well as to the further descriptionand prior art mentioned herein.

Similarly, the nucleotide sequences of the invention may be naturallyoccurring nucleotide sequences or synthetic or semi-synthetic sequences,and may for example be sequences that are isolated by PCR from asuitable naturally occurring template (e.g. DNA or RNA isolated from acell), nucleotide sequences that have been isolated from a library (andin particular, an expression library), nucleotide sequences that havebeen prepared by introducing mutations into a naturally occurringnucleotide sequence (using any suitable technique known per se, such asmismatch PCR), nucleotide sequence that have been prepared by PCR usingoverlapping primers, or nucleotide sequences that have been preparedusing techniques for DNA synthesis known per se.

The amino acid sequence of the invention may in particular a “singlevariable domain” or “single variable domains” (hereinafter “singlevariable domains”). The single variable domains of the invention are anyvariable domain that forms a single antigen binding unit. Generally,such single variable domains will be amino acid sequences thatessentially consist of 4 framework regions (FR1 to FR4 respectively) and3 complementarity determining regions (CDR1 to CDR3 respectively); orany suitable fragment of such an amino acid sequence (which will thenusually contain at least some of the amino acid residues that form atleast one of the CDR's, as further described herein). Such singlevariable domains and fragments are most preferably such that theycomprise an immunoglobulin fold or are capable for forming, undersuitable conditions, an immunoglobulin fold. As such, the singlevariable domain may for example comprise a light chain variable domainsequence (e.g. a V_(L)-sequence) or a suitable fragment thereof; or aheavy chain variable domain sequence (e.g. a V_(H)-sequence or V_(HH)sequence) or a suitable fragment thereof; as long as it is capable offorming a single antigen binding unit (i.e. a functional antigen bindingunit that essentially consists of the single variable domain, such thatthe single antigen binding domain does not need to interact with anothervariable domain to form a functional antigen binding unit, as is forexample the case for the variable domains that are present in forexample conventional antibodies and ScFv fragments that need to interactwith another variable domain—e.g. through a V_(H)/V_(L) interaction—toform a functional antigen binding domain).

For example, the single variable domain may be a domain antibody (or anamino acid sequence that is suitable for use as a domain antibody), asingle domain antibody (or an amino acid sequence that is suitable foruse as a single domain antibody), a “dAb” (or an amino acid sequencethat is suitable for use as a dAb) or a Nanobody™ (as defined herein,and including but not limited to a V_(HH) sequence); other singlevariable domains, or any suitable fragment of any one thereof. For ageneral description of (single) domain antibodies, reference is alsomade to the prior art cited above, as well as to EP 0 368 684. For theterm “dAb's”, reference is for example made to Ward et al. (Nature 1989Oct. 12; 341 (6242): 544-6), to Holt et al., Trends Biotechnol., 2003,21(10:484-490; as well as to for example WO 04/068820, WO 06/030220, WO06/003388 and other published patent applications of Domantis Ltd. Itshould also be noted that, although less preferred in the context of thepresent invention because they are not of mammalian origin, singledomain antibodies or single variable domains can be derived from certainspecies of shark (for example, the so-called “IgNAR domains”, see forexample WO 05/18629).

In particular, the amino acid sequence of the invention may be aNanobody™ or a suitable fragment thereof. [Note: Nanobody™ Nanohodies™and Nanoclone™ are trademarks of Ablynx N.V.] For a further descriptionof V_(HH)'s and Nanobodies, reference is made to the review article byMuyldermans in Reviews in Molecular Biotechnology 74 (2001), 277-302; aswell as to the following patent applications, which are mentioned asgeneral background art: WO 94/04678, WO 95/04079 and WO 96/34103 of theVrije Universiteit Brussel; WO 94/25591, WO 99/37681, WO 00/40968, WO00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231 and WO02/48193 of Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO03/054016 and WO 03/055527 of the Vlaams Instituut voor Biotechnologie(VIB); WO 03/050531 of Algonomics N.V. and Ablynx N.V.; WO 01/90190 bythe National Research Council of Canada; WO 03/025020 (=EP 1 433 793) bythe Institute of Antibodies; as well as WO 04/041867, WO 04/041862, WO04/041865, WO 04/041863, WO 04/062551, WO 05/044858, WO 06/40153, WO06/079372, WO 06/122786, WO 06/122787 and WO 06/122825, by Ablynx N.V.and the further published patent applications by Ablynx N.V. Referenceis also made to the further prior art mentioned in these applications,and in particular to the list of references mentioned on pages 41-43 ofthe International application WO 06/040153, which list and referencesare incorporated herein by reference. As described in these references,Generally, Nanobodies (in particular V_(HH) sequences and partiallyhumanized Nanobodies) can in particular be characterized by the presenceof one or more “Hallmark residues” in one or more of the frameworksequences.

The amino acid sequence of the invention may in particular be a domainantibody (or an amino acid sequence that is suitable for use as a domainantibody), a single domain antibody (or an amino acid sequence that issuitable for use as a single domain antibody), a “dAb” (or an amino acidsequence that is suitable for use as a dAb) or a Nanobody™ (as definedherein, and including but not limited to a V_(HH) sequence); othersingle variable domains, or any suitable fragment of any one thereof.For a general description of (single) domain antibodies, reference isalso made to the prior art cited above, as well as to EP 0 368 684. Forthe term “dAb's”, reference is for example made to Ward et al. (Nature1989 Oct. 12; 341 (6242): 544-6), to Holt et al., Trends Biotechnol.,2003, 21(11):484-490; as well as to for example WO 06/030220, WO06/003388 and other published patent applications of Domantis Ltd. Itshould also be noted that, although less preferred in the context of thepresent invention because they are not of mammalian origin, singledomain antibodies or single variable domains can be derived from certainspecies of shark (for example, the so-called “IgNAR domains”, see forexample WO 05/18629).

In particular, the amino acid sequence of the invention may be aNanobody® (as defined herein) or a suitable fragment thereof [Note:Nanobody®; Nanobodies® and Nanoclone® are trademarks of Ablynx N.V.]Such Nanobodies directed against Tie1, Tie2, Ang1, Ang2, Ang3, Ang4,Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6 will also bereferred to herein as “Nanobodies of the invention”.

For a general description of Nanobodies, reference is made to thefurther description below, as well as to the prior art cited herein. Inthis respect, it should however be noted that this description and theprior art mainly described Nanobodies of the so-called “V_(H)3 class”(i.e. Nanobodies with a high degree of sequence homology to humangermline sequences of the V_(H)3 class such as DP-47, DP-51 or DP-29),which Nanobodies form a preferred aspect of this invention. It shouldhowever be noted that the invention in its broadest sense generallycovers any type of Nanobody directed against Tie1, Tie2, Ang1, Ang2,Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, andfor example also covers the Nanobodies belonging to the so-called“V_(H)4 class” (i.e. Nanobodies with a high degree of sequence homologyto human germline sequences of the V_(H)4 class such as DP-78), as forexample described in the U.S. provisional application 60/792,279 byAblynx N.V. entitled “DP-78-like Nanobodies” filed on Apr. 14, 2006.

Generally, Nanobodies (in particular V_(HH) sequences and partiallyhumanized Nanobodies) can in particular be characterized by the presenceof one or more “Hallmark residues” (as described herein) in one or moreof the framework sequences (again as further described herein).

Thus, generally, a Nanobody can be defined as an amino acid sequencewith the (general) structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which one or more of the Hallmark residuesare as further defined herein.

In particular, a Nanobody can be an amino acid sequence with the(general) structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which the framework sequences are as furtherdefined herein.

More in particular, a Nanobody can be an amino acid sequence with the(general) structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   i) preferably one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    A-3 below;    and in which:-   ii) said amino acid sequence has at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 1 to    22, in which for the purposes of determining the degree of amino    acid identity, the amino acid residues that form the CDR sequences    (indicated with X in the sequences of SEQ ID NO's: 1 to 22) are    disregarded.

In these Nanobodies, the CDR sequences are generally as further definedherein.

Thus, the invention also relates to such Nanobodies that can bind to (asdefined herein) and/or are directed against Tie1, Tie2, Ang1, Ang2,Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, tosuitable fragments thereof, as well as to polypeptides that comprise oressentially consist of one or more of such Nanobodies and/or suitablefragments.

SEQ ID NO's 455 to 501 give the amino acid sequences of a number ofV_(HH) sequences that have been raised against Tie2, Ang1, Ang2, andAng4.

Accordingly, some particularly preferred Nanobodies of the invention areNanobodies which can bind (as further defined herein) to and/or aredirected against to Tie2, Ang1, Ang2, Ang4 or Angptl4 and which:

-   i) have 80% amino acid identity with at least one of the amino acid    sequences of SEQ ID NO's: 455 to 501, in which for the purposes of    determining the degree of amino acid identity, the amino acid    residues that form the CDR sequences are disregarded. In this    respect, reference is also made to Table A-1, which lists the    framework 1 sequences (SEQ ID NO's: 126 to 172), framework 2    sequences (SEQ ID NO's: 220 to 266), framework 3 sequences (SEQ ID    NO's: 314 to 360) and framework 4 sequences (SEQ ID NO's: 408    to 454) of the Nanobodies of SEQ ID NO's: 455 to 501 (with respect    to the amino acid residues at positions 1 to 4 and 27 to 30 of the    framework 1 sequences, reference is also made to the comments made    below. Thus, for determining the degree of amino acid identity,    these residues are preferably disregarded);    and in which:-   ii) preferably one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    A-3 below.

In these Nanobodies, the CDR sequences are generally as further definedherein.

Again, such Nanobodies may be derived in any suitable manner and fromany suitable source, and may for example be naturally occurring V_(HH)sequences (i.e. from a suitable species of Camelid) or synthetic orsemi-synthetic amino acid sequences, including but not limited to“humanized” (as defined herein) Nanobodies, “camelized” (as definedherein) immunoglobulin sequences (and in particular camelized heavychain variable domain sequences), as well as Nanobodies that have beenobtained by techniques such as affinity maturation (for example,starting from synthetic, random or naturally occurring immunoglobulinsequences), CDR grafting, veneering, combining fragments derived fromdifferent immunoglobulin sequences, PCR assembly using overlappingprimers, and similar techniques for engineering immunoglobulin sequenceswell known to the skilled person; or any suitable combination of any ofthe foregoing as further described herein. Also, when a Nanobodycomprises a V_(HH) sequence, said Nanobody may be suitably humanized, asfurther described herein, so as to provide one or more further(partially or fully) humanized Nanobodies of the invention. Similarly,when a Nanobody comprises a synthetic or semi-synthetic sequence (suchas a partially humanized sequence), said Nanobody may optionally befurther suitably humanized, again as described herein, again so as toprovide one or more further (partially or fully) humanized Nanobodies ofthe invention.

In particular, humanized Nanobodies may be amino acid sequences that areas generally defined for Nanobodies in the previous paragraphs, but inwhich at least one amino acid residue is present (and in particular, inat least one of the framework residues) that is and/or that correspondsto a humanizing substitution (as defined herein). Some preferred, butnon-limiting humanizing substitutions (and suitable combinationsthereof) will become clear to the skilled person based on the disclosureherein. In addition, or alternatively, other potentially usefulhumanizing substitutions can be ascertained by comparing the sequence ofthe framework regions of a naturally occurring V_(HH) sequence with thecorresponding framework sequence of one or more closely related humanV_(H) sequences, after which one or more of the potentially usefulhumanizing substitutions (or combinations thereof) thus determined canbe introduced into said V_(HH)-sequence (in any manner known per se, asfurther described herein) and the resulting humanized V_(HH) sequencescan be tested for affinity for the target, for stability, for ease andlevel of expression, and/or for other desired properties. In this way,by means of a limited degree of trial and error, other suitablehumanizing substitutions (or suitable combinations thereof) can bedetermined by the skilled person based on the disclosure herein. Also,based on the foregoing, (the framework regions of) a Nanobody may bepartially humanized or fully humanized.

Some particularly preferred humanized Nanobodies of the invention arehumanized variants of the Nanobodies of SEQ ID NO's: 455 to 501, ofwhich the amino acid sequences of SEQ ID NO's: 455 to 457, 459, 460, 464to 469 are some especially preferred examples.

Thus, some other preferred Nanobodies of the invention are Nanobodieswhich can block (as further defined herein) the Ang1/Tie2 or Ang2/Tie2interaction and which:

-   i) preferably one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    A-3 below.

According to another specific aspect of the invention, the inventionprovides a number of stretches of amino acid residues (i.e. smallpeptides) that are particularly suited for binding to Tie1, Tie2, Ang1,Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6. These stretches of amino acid residues may be present in,and/or may be incorporated into an amino acid sequence of the invention,in particular in such a way that they form (part of) the antigen bindingsite of an amino acid sequence of the invention. As these stretches ofamino acid residues were first generated as CDR sequences of heavy chainantibodies or V_(HH) sequences that were raised against Tie1, Tie2,Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6 (or may be based on and/or derived from such CDR sequences, asfurther described herein), they will also generally be referred toherein as “CDR sequences” (i.e. as CDR1 sequences, CDR2 sequences andCDR3 sequences, respectively). It should however be noted that theinvention in its broadest sense is not limited to a specific structuralrole or function that these stretches of amino acid residues may have inan amino acid sequence of the invention, as long as these stretches ofamino acid residues allow the amino acid sequence of the invention tobind to Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl 1, Angptl2, Angptl3,Angptl4, Angptl5, or Angptl6. Thus, generally, the invention in itsbroadest sense comprises any amino acid sequence that is capable ofbinding to Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,Angptl3, Angptl4, Angptl5, or Angptl6 and that comprises one or more CDRsequences as described herein, and in particular a suitable combinationof two or more such CDR sequences, that are suitably linked to eachother via one or more further amino acid sequences, such that the entireamino acid sequence forms a binding domain and/or binding unit that iscapable of binding to Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1,Angptl2, Angptl3, Angptl4. Angptl5, or Angptl6. It should however alsobe noted that the presence of only one such CDR sequence in an aminoacid sequence of the invention may by itself already be sufficient toprovide an amino acid sequence of the invention that is capable ofbinding to Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,Angptl3, Angptl4, Angptl5, or Angptl6; reference is for example againmade to the so-called “Expedite fragments” described in WO 03/050531.

Thus, in another specific, but non-limiting aspect, the amino acidsequence of the invention may be an amino acid sequence that comprisesat least one amino acid sequence that is chosen from the groupconsisting of the CDR1 sequences, CDR2 sequences and CDR3 sequences thatare described herein (or any suitable combination thereof). Inparticular, an amino acid sequence of the invention may be an amino acidsequence that comprises at least one antigen binding site, wherein saidantigen binding site comprises at least one amino acid sequence that ischosen from the group consisting of the CDR1 sequences, CDR2 sequencesand CDR3 sequences that are described herein (or any suitablecombination thereof).

Generally, in this aspect of the invention, the amino acid sequence ofthe invention may be any amino acid sequence that comprises at least onestretch of amino acid residues, in which said stretch of amino acidresidues has an amino acid sequence that corresponds to the sequence ofat least one of the CDR sequences described herein. Such an amino acidsequence may or may not comprise an immunoglobulin fold. For example,and without limitation, such an amino acid sequence may be a suitablefragment of an immunoglobulin sequence that comprises at least one suchCDR sequence, but that is not large enough to form a (complete)immunoglobulin fold (reference is for example again made to the“Expedite fragments” described in WO 03/050531). Alternatively, such anamino acid sequence may be a suitable “protein scaffold” that comprisesleast one stretch of amino acid residues that corresponds to such a CDRsequence (i.e. as part of its antigen binding site). Suitable scaffoldsfor presenting amino acid sequences will be clear to the skilled person,and for example comprise, without limitation, to binding scaffolds basedon or derived from immunoglobulins (i.e. other than the immunoglobulinsequences already described herein), protein scaffolds derived fromprotein A domains (such as Affibodies™), tendamistat, fibronectin,lipocalin, CTLA-4, T-cell receptors, designed ankyrin repeats, avimersand PDZ domains (Binz et al., Nat. Biotech 2005. Vol 23:1257), andbinding moieties based on DNA or RNA including but not limited to DNA orRNA aptamers (Ulrich et al., Comb Chem High Throughput Screen 20069(8):619-32).

Again, any amino acid sequence of the invention that comprises one ormore of these CDR sequences is preferably such that it can specificallybind (as defined herein) to Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1,Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, and more in particularsuch that it can bind to Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1,Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6 with an affinity(suitably measured and/or expressed as a K_(D)-value (actual orapparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or ak_(off)-rate, or alternatively as an IC₅₀ value, as further describedherein), that is as defined herein.

More in particular, the amino acid sequences according to this aspect ofthe invention may be any amino acid sequence that comprises at least oneantigen binding site, wherein said antigen binding site comprises atleast two amino acid sequences that are chosen from the group consistingof the CDR1 sequences described herein, the CDR2 sequences describedherein and the CDR3 sequences described herein, such that (i) when thefirst amino acid sequence is chosen from the CDR1 sequences describedherein, the second amino acid sequence is chosen from the CDR2 sequencesdescribed herein or the CDR3 sequences described herein; (ii) when thefirst amino acid sequence is chosen from the CDR2 sequences describedherein, the second amino acid sequence is chosen from the CDR1 sequencesdescribed herein or the CDR3 sequences described herein; or (iii) whenthe first amino acid sequence is chosen from the CDR3 sequencesdescribed herein, the second amino acid sequence is chosen from the CDR1sequences described herein or the CDR3 sequences described herein.

Even more in particular, the amino acid sequences of the invention maybe amino acid sequences that comprise at least one antigen binding site,wherein said antigen binding site comprises at least three amino acidsequences that are chosen from the group consisting of the CDR1sequences described herein, the CDR2 sequences described herein and theCDR3 sequences described herein, such that the first amino acid sequenceis chosen from the CDR1 sequences described herein, the second aminoacid sequence is chosen from the CDR2 sequences described herein, andthe third amino acid sequence is chosen from the CDR3 sequencesdescribed herein. Preferred combinations of CDR1, CDR2 and CDR3sequences will become clear from the further description herein. As willbe clear to the skilled person, such an amino acid sequence ispreferably an immunoglobulin sequence (as further described herein), butit may for example also be any other amino acid sequence that comprisesa suitable scaffold for presenting said CDR sequences.

Thus, in one specific, but non-limiting aspect, the invention relates toan amino acid sequence directed against Tie1, Tie2, Ang1, Ang2, Ang3,Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, thatcomprises one or more stretches of amino acid residues chosen from thegroup consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 173 to 219;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 173 to    219;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 173 to    219;-   d) the amino acid sequences of SEQ ID NO's: 267 to 313;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 267 to    313;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 267 to    313;-   g) the amino acid sequences of SEQ ID NO's: 361 to 454;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 361 to    454;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 361 to    454;    or any suitable combination thereof.

When an amino acid sequence of the invention contains one or more aminoacid sequences according to b) and/or c):

-   i) any amino acid substitution in such an amino acid sequence    according to b) and/or c) is preferably, and compared to the    corresponding amino acid sequence according to a), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to b) and/or c) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to a);    and/or-   iii) the amino acid sequence according to b) and/or c) may be an    amino acid sequence that is derived from an amino acid sequence    according to a) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

Similarly, when an amino acid sequence of the invention contains one ormore amino acid sequences according to e) and/or f):

-   i) any amino acid substitution in such an amino acid sequence    according to e) and/or f) is preferably, and compared to the    corresponding amino acid sequence according to d), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to e) and/or f) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to d);    and/or-   iii) the amino acid sequence according to e) and/or f) may be an    amino acid sequence that is derived from an amino acid sequence    according to d) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

Also, similarly, when an amino acid sequence of the invention containsone or more amino acid sequences according to h) and/or i):

-   i) any amino acid substitution in such an amino acid sequence    according to h) and/or i) is preferably, and compared to the    corresponding amino acid sequence according to g), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to h) and/or i) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to g);    and/or-   iii) the amino acid sequence according to h) and/or i) may be an    amino acid sequence that is derived from an amino acid sequence    according to g) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

It should be understood that the last preceding paragraphs alsogenerally apply to any amino acid sequences of the invention thatcomprise one or more amino acid sequences according to b), c), e), f),h) or i), respectively.

In this specific aspect, the amino acid sequence preferably comprisesone or more stretches of amino acid residues chosen from the groupconsisting of:

i) the amino acid sequences of SEQ ID NO's: 173 to 219;ii) the amino acid sequences of SEQ ID NO's: 267 to 313; andiii) the amino acid sequences of SEQ ID NO's: 361 to 454;or any suitable combination thereof.

Also, preferably, in such an amino acid sequence, at least one of saidstretches of amino acid residues forms part of the antigen binding sitefor binding against Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1,Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6.

In a more specific, but again non-limiting aspect, the invention relatesto an amino acid sequence directed against Tie1, Tie2, Ang1, Ang2, Ang3,Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, thatcomprises two or more stretches of amino acid residues chosen from thegroup consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 173 to 219;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 173 to    219;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 173 to    219;-   d) the amino acid sequences of SEQ ID NO's: 267 to 313;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 267 to    313;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 267 to    313;-   g) the amino acid sequences of SEQ ID NO's: 361 to 454;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 361 to    454;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 361 to    454;    such that (i) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences according to a), b)    or c), the second stretch of amino acid residues corresponds to one    of the amino acid sequences according to d), e), f), g), h) or    i); (ii) when the first stretch of amino acid residues corresponds    to one of the amino acid sequences according to d), e) or f), the    second stretch of amino acid residues corresponds to one of the    amino acid sequences according to a), b), c), g), h) or i); or (iii)    when the first stretch of amino acid residues corresponds to one of    the amino acid sequences according to g), h) or i), the second    stretch of amino acid residues corresponds to one of the amino acid    sequences according to a), b), c), d), e) or f).

In this specific aspect, the amino acid sequence preferably comprisestwo or more stretches of amino acid residues chosen from the groupconsisting of:

i) the amino acid sequences of SEQ ID NO's: 173 to 219;ii) the amino acid sequences of SEQ ID NO's: 267 to 313; andiii) the amino acid sequences of SEQ ID NO's: 361 to 454;such that, (i) when the first stretch of amino acid residues correspondsto one of the amino acid sequences of SEQ ID NO's: 173 to 219, thesecond stretch of amino acid residues corresponds to one of the aminoacid sequences of SEQ ID NO's: 267 to 313 or of SEQ ID NO's: 361 to 454;(ii) when the first stretch of amino acid residues corresponds to one ofthe amino acid sequences of SEQ ID NO's: 267 to 313, the second stretchof amino acid residues corresponds to one of the amino acid sequences ofSEQ ID NO's: 173 to 219 or of SEQ ID NO's: 361 to 454; or (iii) when thefirst stretch of amino acid residues corresponds to one of the aminoacid sequences of SEQ ID NO's: 361 to 454, the second stretch of aminoacid residues corresponds to one of the amino acid sequences of SEQ IDNO's: 173 to 219 or of SEQ ID NO's: 267 to 313.

Also, in such an amino acid sequence, the at least two stretches ofamino acid residues again preferably form part of the antigen bindingsite for binding against Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1,Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6.

In an even more specific, but non-limiting aspect, the invention relatesto an amino acid sequence directed against Tie1, Tie2, Ang1, Ang2, Ang3,Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, thatcomprises three or more stretches of amino acid residues, in which thefirst stretch of amino acid residues is chosen from the group consistingof:

-   a) the amino acid sequences of SEQ ID NO's: 173 to 219;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 173 to    219;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 173 to    219; the second stretch of amino acid residues is chosen from the    group consisting of:-   d) the amino acid sequences of SEQ ID NO's: 267 to 313;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 267 to    313;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 267 to    313; and the third stretch of amino acid residues is chosen from the    group consisting of:-   g) the amino acid sequences of SEQ ID NO's: 361 to 454;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 361 to    454;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 361 to    454.

Preferably, in this specific aspect, the first stretch of amino acidresidues is chosen from the group consisting of the amino acid sequencesof SEQ ID NO's: 173 to 219; the second stretch of amino acid residues ischosen from the group consisting of the amino acid sequences of SEQ IDNO's: 267 to 313; and the third stretch of amino acid residues is chosenfrom the group consisting of the amino acid sequences of SEQ ID NO's:361 to 454.

Again, preferably, in such an amino acid sequence, the at least threestretches of amino acid residues forms part of the antigen binding sitefor binding against Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1,Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6.

Preferred combinations of such stretches of amino acid sequences willbecome clear from the further disclosure herein.

Preferably, in such amino acid sequences the CDR sequences have at least70% amino acid identity, preferably at least 80% amino acid identity,more preferably at least 90% amino acid identity, such as 95% amino acididentity or more or even essentially 100% amino acid identity with theCDR sequences of at least one of the amino acid sequences of SEQ IDNO's: 455 to 501. This degree of amino acid identity can for example bedetermined by determining the degree of amino acid identity (in a mannerdescribed herein) between said amino acid sequence and one or more ofthe sequences of SEQ ID NO's: 455 to 501, in which the amino acidresidues that form the framework regions are disregarded. Also, suchamino acid sequences of the invention can be as further describedherein.

Also, such amino acid sequences are preferably such that they canspecifically bind (as defined herein) to Tie1, Tie2, Ang1, Ang2, Ang3,Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6; and morein particular bind to Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1,Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6 with an affinity(suitably measured and/or expressed as a K_(D)-value (actual orapparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or ak_(off)-rate, or alternatively as an IC₅₀ value, as further describedherein) that is as defined herein.

When the amino acid sequence of the invention essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), the amino acidsequence of the invention is preferably such that:

CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 173 to 219;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 173 to    219;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 173 to    219;    and/or

CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 267 to 313;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 267 to    313;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 267 to    313;    and/or

CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 361 to 454;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 361 to    454;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 361 to    454.

In particular, such an amino acid sequence of the invention may be suchthat CDR1 is chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 173 to 219; and/or CDR2 is chosen from thegroup consisting of the amino acid sequences of SEQ ID NO's: 267 to 313,and/or CDR3 is chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 361 to 454.

In particular, when the amino acid sequence of the invention essentiallyconsists of 4 framework regions (FR1 to FR4, respectively) and 3complementarity determining regions (CDR1 to CDR3, respectively), theamino acid sequence of the invention is preferably such that:

CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 173 to 219;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 173 to    219;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 173 to    219;    and

CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 267 to 313;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 267 to    313;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 267 to    313;    and

CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 267 to 313;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 267 to    313;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 267 to    313; or any suitable fragment of such an amino acid sequence

In particular, such an amino acid sequence of the invention may be suchthat CDR1 is chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 173 to 219; and CDR2 is chosen from the groupconsisting of the amino acid sequences of SEQ ID NO's: 267 to 313; andCDR3 is chosen from the group consisting of the amino acid sequences ofSEQ ID NO's: 361 to 454.

Again, preferred combinations of CDR sequences will become clear fromthe further description herein.

Also, such amino acid sequences are preferably such that they canspecifically bind (as defined herein) to Tie1, Tie2, Ang1, Ang2, Ang3,Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6; and morein particular bind to Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1,Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6 with an affinity(suitably measured and/or expressed as a K_(D)-value (actual orapparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or ak_(off)-rate, or alternatively as an IC₅₀ value, as further describedherein) that is as defined herein.

In one preferred, but non-limiting aspect, the invention relates to anamino acid sequence that essentially consists of 4 framework regions(FR1 to FR4, respectively) and 3 complementarity determining regions(CDR1 to CDR3, respectively), in which the CDR sequences of said aminoacid sequence have at least 70% amino acid identity, preferably at least80% amino acid identity, more preferably at least 90% amino acididentity, such as 95% amino acid identity or more or even essentially100% amino acid identity with the CDR sequences of at least one of theamino acid sequences of SEQ ID NO's: 455 to 501. This degree of aminoacid identity can for example be determined by determining the degree ofamino acid identity (in a manner described herein) between said aminoacid sequence and one or more of the sequences of SEQ ID NO's: 455 to501, in which the amino acid residues that form the framework regionsare disregarded. Such amino acid sequences of the invention can be asfurther described herein.

In one preferred, but non-limiting aspect, the invention relates to anamino acid sequence that binds to Tie2, blocks interaction with Ang1 andessentially consists of 4 framework regions (FR1 to FR4, as describedabove, e.g. humanized framework regions FR1, FR2, FR3 or FR4 of any FR1,FR2, FR3 or FR4 as shown in Table A-1 (or preferably any correspondingFR for Tie2 binders with SEQ ID NO's: 455 to 457, 459, or 460) or anyFR1, FR2, FR3 or FR4 as shown in Table A-1 (or preferably anycorresponding FR for Tie2 binders with SEQ ID NO's: 455 to 457, 459, or460)) and 3 complementarity determining regions (CDR1 to CDR3,respectively), in which the CDR sequences of said amino acid sequencehave at least 70% amino acid identity, preferably at least 80% aminoacid identity, more preferably at least 90% amino acid identity, such as95% amino acid identity or more or even essentially 100% amino acididentity with the CDR sequences of at least one of the amino acidsequences of SEQ ID NO's: 455 to 457, 459, or 460. This degree of aminoacid identity can for example be determined by determining the degree ofamino acid identity (in a manner described herein) between said aminoacid sequence and one or more of the sequences of SEQ ID NO's: 455 to457, 459, or 460, in which the amino acid residues that form theframework regions are disregarded. Such amino acid sequences of theinvention can be as further described herein.

In one preferred, but non-limiting aspect, the invention relates to anamino acid sequence that binds to Ang2, blocks interaction with Tie2 andessentially consists of 4 framework regions (FR1 to FR4, as describedabove, e.g. humanized framework regions FR1, FR2, FR3 or FR4 of any FR1,FR2, FR3 or FR4 as shown in Table A-1 (or preferably any correspondingFR for Ang2 binders with SEQ ID NO's: 464 to 469) or any FR1, FR2, FR3or FR4 as shown in Table A-1 (or preferably any corresponding FR forAng2 binders with SEQ ID NO's: 464 to 469)) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which the CDRsequences of said amino acid sequence have at least 70% amino acididentity, preferably at least 80% amino acid identity, more preferablyat least 90% amino acid identity, such as 95% amino acid identity ormore or even essentially 100% amino acid identity with the CDR sequencesof at least one of the amino acid sequences of SEQ ID NO's: 464 to 469.This degree of amino acid identity can for example be determined bydetermining the degree of amino acid identity (in a manner describedherein) between said amino acid sequence and one or more of thesequences of SEQ ID NO's: 464 to 469, in which the amino acid residuesthat form the framework regions are disregarded. Such amino acidsequences of the invention can be as further described herein.

In one preferred, but non-limiting aspect, the invention relates to anamino acid sequence that binds to Tie2, blocks interaction with Ang1 andessentially consists of 4 framework regions (FR1 to FR4 as described inSEQ ID NO: 455, or a humanized framework thereof) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which the CDRsequences of said amino acid sequence have at least 70% amino acididentity, preferably at least 80% amino acid identity, more preferablyat least 90% amino acid identity, such as 95% amino acid identity ormore or even essentially 100% amino acid identity with the CDR sequencesof at least one of the amino acid sequences of SEQ ID NO's: 455. Thisdegree of amino acid identity can for example be determined bydetermining the degree of amino acid identity (in a manner describedherein) between said amino acid sequence and the sequence of SEQ IDNO's: 455, in which the amino acid residues that form the frameworkregions are disregarded. Such amino acid sequences of the invention canbe as further described herein.

In one preferred, but non-limiting aspect, the invention relates to anamino acid sequence that binds to Tie2, blocks interaction with Ang1 andessentially consists of 4 framework regions (FR1 to FR4 as described inSEQ ID NO: 456, or a humanized framework thereof) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which the CDRsequences of said amino acid sequence have at least 70% amino acididentity, preferably at least 80% amino acid identity, more preferablyat least 90% amino acid identity, such as 95% amino acid identity ormore or even essentially 100% amino acid identity with the CDR sequencesof at least one of the amino acid sequences of SEQ ID NO's: 456. Thisdegree of amino acid identity can for example be determined bydetermining the degree of amino acid identity (in a manner describedherein) between said amino acid sequence and the sequence of SEQ IDNO's: 456, in which the amino acid residues that form the frameworkregions are disregarded. Such amino acid sequences of the invention canbe as further described herein.

In one preferred, but non-limiting aspect, the invention relates to anamino acid sequence that binds to Tie2, blocks interaction with Ang1 andessentially consists of 4 framework regions (FR1 to FR4 as described inSEQ ID NO: 457, or a humanized framework thereof) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which the CDRsequences of said amino acid sequence have at least 70% amino acididentity, preferably at least 80% amino acid identity, more preferablyat least 90% amino acid identity, such as 95% amino acid identity ormore or even essentially 100% amino acid identity with the CDR sequencesof at least one of the amino acid sequences of SEQ ID NO's: 457. Thisdegree of amino acid identity can for example be determined bydetermining the degree of amino acid identity (in a manner describedherein) between said amino acid sequence and the sequence of SEQ IDNO's: 457, in which the amino acid residues that form the frameworkregions are disregarded. Such amino acid sequences of the invention canbe as further described herein.

In one preferred, but non-limiting aspect, the invention relates to anamino acid sequence that binds to Tie2, blocks interaction with Ang1 andessentially consists of 4 framework regions (FR1 to FR4 as described inSEQ ID NO: 459, or a humanized framework thereof) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which the CDRsequences of said amino acid sequence have at least 70% amino acididentity, preferably at least 80% amino acid identity, more preferablyat least 90% amino acid identity, such as 95% amino acid identity ormore or even essentially 100% amino acid identity with the CDR sequencesof at least one of the amino acid sequences of SEQ ID NO's: 459. Thisdegree of amino acid identity can for example be determined bydetermining the degree of amino acid identity (in a manner describedherein) between said amino acid sequence and the sequence of SEQ IDNO's: 459, in which the amino acid residues that form the frameworkregions are disregarded. Such amino acid sequences of the invention canbe as further described herein.

In one preferred, but non-limiting aspect, the invention relates to anamino acid sequence that binds to Tie2, blocks interaction with Ang1 andessentially consists of 4 framework regions (FR1 to FR4 as described inSEQ ID NO: 460, or a humanized framework thereof) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which the CDRsequences of said amino acid sequence have at least 70% amino acididentity, preferably at least 80% amino acid identity, more preferablyat least 90% amino acid identity, such as 95% amino acid identity ormore or even essentially 100% amino acid identity with the CDR sequencesof at least one of the amino acid sequences of SEQ ID NO's: 460. Thisdegree of amino acid identity can for example be determined bydetermining the degree of amino acid identity (in a manner describedherein) between said amino acid sequence and the sequence of SEQ IDNO's: 460, in which the amino acid residues that form the frameworkregions are disregarded. Such amino acid sequences of the invention canbe as further described herein.

In one preferred, but non-limiting aspect, the invention relates to anamino acid sequence that binds to Ang2, blocks interaction with Tie2 andessentially consists of 4 framework regions (FR1 to FR4 as described inSEQ ID NO: 464, or a humanized framework thereof) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which the CDRsequences of said amino acid sequence have at least 70% amino acididentity, preferably at least 80% amino acid identity, more preferablyat least 90% amino acid identity, such as 95% amino acid identity ormore or even essentially 100% amino acid identity with the CDR sequencesof at least one of the amino acid sequences of SEQ ID NO's: 464. Thisdegree of amino acid identity can for example be determined bydetermining the degree of amino acid identity (in a manner describedherein) between said amino acid sequence and the sequence of SEQ IDNO's: 464, in which the amino acid residues that form the frameworkregions are disregarded. Such amino acid sequences of the invention canbe as further described herein.

In one preferred, but non-limiting aspect, the invention relates to anamino acid sequence that binds to Ang2, blocks interaction with Tie2 andessentially consists of 4 framework regions (FR1 to FR4 as described inSEQ ID NO: 465, or a humanized framework thereof) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which the CDRsequences of said amino acid sequence have at least 70% amino acididentity, preferably at least 80% amino acid identity, more preferablyat least 90% amino acid identity, such as 95% amino acid identity ormore or even essentially 100% amino acid identity with the CDR sequencesof at least one of the amino acid sequences of SEQ ID NO's: 465. Thisdegree of amino acid identity can for example be determined bydetermining the degree of amino acid identity (in a manner describedherein) between said amino acid sequence and the sequence of SEQ IDNO's: 465, in which the amino acid residues that form the frameworkregions are disregarded. Such amino acid sequences of the invention canbe as further described herein.

In one preferred, but non-limiting aspect, the invention relates to anamino acid sequence that binds to Ang2, blocks interaction with Tie2 andessentially consists of 4 framework regions (FR1 to FR4 as described inSEQ ID NO: 466, or a humanized framework thereof) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which the CDRsequences of said amino acid sequence have at least 70% amino acididentity, preferably at least 80% amino acid identity, more preferablyat least 90% amino acid identity, such as 95% amino acid identity ormore or even essentially 100% amino acid identity with the CDR sequencesof at least one of the amino acid sequences of SEQ ID NO's: 466. Thisdegree of amino acid identity can for example be determined bydetermining the degree of amino acid identity (in a manner describedherein) between said amino acid sequence and the sequence of SEQ IDNO's: 466, in which the amino acid residues that form the frameworkregions are disregarded. Such amino acid sequences of the invention canbe as further described herein.

In one preferred, but non-limiting aspect, the invention relates to anamino acid sequence that binds to Ang2, blocks interaction with Tie2 andessentially consists of 4 framework regions (FR1 to FR4 as described inSEQ ID NO: 467, or a humanized framework thereof) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which the CDRsequences of said amino acid sequence have at least 70%/amino acididentity, preferably at least 80% amino acid identity, more preferablyat least 90% amino acid identity, such as 95% amino acid identity ormore or even essentially 100% amino acid identity with the CDR sequencesof at least one of the amino acid sequences of SEQ ID NO's: 467. Thisdegree of amino acid identity can for example be determined bydetermining the degree of amino acid identity (in a manner describedherein) between said amino acid sequence and the sequence of SEQ IDNO's: 467, in which the amino acid residues that form the frameworkregions are disregarded. Such amino acid sequences of the invention canbe as further described herein.

In one preferred, but non-limiting aspect, the invention relates to anamino acid sequence that binds to Ang2, blocks interaction with Tie2 andessentially consists of 4 framework regions (FR1 to FR4 as described inSEQ ID NO: 468, or a humanized framework thereof) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which the CDRsequences of said amino acid sequence have at least 70% amino acididentity, preferably at least 80% amino acid identity, more preferablyat least 90% amino acid identity, such as 95% amino acid identity ormore or even essentially 100% amino acid identity with the CDR sequencesof at least one of the amino acid sequences of SEQ ID NO's: 468. Thisdegree of amino acid identity can for example be determined bydetermining the degree of amino acid identity (in a manner describedherein) between said amino acid sequence and the sequence of SEQ IDNO's: 468, in which the amino acid residues that form the frameworkregions are disregarded. Such amino acid sequences of the invention canbe as further described herein.

In one preferred, but non-limiting aspect, the invention relates to anamino acid sequence that binds to Ang2, blocks interaction with Tie2 andessentially consists of 4 framework regions (FR1 to FR4 as described inSEQ ID NO: 469, or a humanized framework thereof) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which the CDRsequences of said amino acid sequence have at least 70% amino acididentity, preferably at least 80% amino acid identity, more preferablyat least 90% amino acid identity, such as 95% amino acid identity ormore or even essentially 100% amino acid identity with the CDR sequencesof at least one of the amino acid sequences of SEQ ID NO's: 469. Thisdegree of amino acid identity can for example be determined bydetermining the degree of amino acid identity (in a manner describedherein) between said amino acid sequence and the sequence of SEQ IDNO's: 469, in which the amino acid residues that form the frameworkregions are disregarded. Such amino acid sequences of the invention canbe as further described herein.

In such an amino acid sequence of the invention, the framework sequencesmay be any suitable framework sequences, and examples of suitableframework sequences will be clear to the skilled person, for example onthe basis the standard handbooks and the further disclosure and priorart mentioned herein.

The framework sequences are preferably (a suitable combination of)immunoglobulin framework sequences or framework sequences that have beenderived from immunoglobulin framework sequences (for example, byhumanization or camelization). For example, the framework sequences maybe framework sequences derived from a light chain variable domain (e.g.a V_(L)-sequence) and/or from a heavy chain variable domain (e.g. aV_(H)-sequence). In one particularly preferred aspect, the frameworksequences are either framework sequences that have been derived from aV_(HH)-sequence (in which said framework sequences may optionally havebeen partially or fully humanized) or are conventional V_(H) sequencesthat have been camelized (as defined herein).

The framework sequences are preferably such that the amino acid sequenceof the invention is a domain antibody (or an amino acid sequence that issuitable for use as a domain antibody); is a single domain antibody (oran amino acid sequence that is suitable for use as a single domainantibody); is a “dAb” (or an amino acid sequence that is suitable foruse as a dAb); or is a Nanobody™ (including but not limited to V_(HH)sequence). Again, suitable framework sequences will be clear to theskilled person, for example on the basis the standard handbooks and thefurther disclosure and prior art mentioned herein.

In particular, the framework sequences present in the amino acidsequences of the invention may contain one or more of Hallmark residues(as defined herein), such that the amino acid sequence of the inventionis a Nanobody™. Some preferred, but non-limiting examples of (suitablecombinations of) such framework sequences will become clear from thefurther disclosure herein.

Again, as generally described herein for the amino acid sequences of theinvention, it is also possible to use suitable fragments (orcombinations of fragments) of any of the foregoing, such as fragmentsthat contain one or more CDR sequences, suitably flanked by and/orlinked via one or more framework sequences (for example, in the sameorder as these CDR's and framework sequences may occur in the full-sizedimmunoglobulin sequence from which the fragment has been derived). Suchfragments may also again be such that they comprise or can form animmunoglobulin fold, or alternatively be such that they do not compriseor cannot form an immunoglobulin fold.

In one specific aspect, such a fragment comprises a single CDR sequenceas described herein (and in particular a CDR3 sequence), that is flankedon each side by (part of) a framework sequence (and in particular, partof the framework sequence(s) that, in the immunoglobulin sequence fromwhich the fragment is derived, are adjacent to said CDR sequence. Forexample, a CDR3 sequence may be preceded by (part of) a FR3 sequence andfollowed by (part of) a FR4 sequence). Such a fragment may also containa disulphide bridge, and in particular a disulphide bridge that linksthe two framework regions that precede and follow the CDR sequence,respectively (for the purpose of forming such a disulphide bridge,cysteine residues that naturally occur in said framework regions may beused, or alternatively cysteine residues may be synthetically added toor introduced into said framework regions).

For a further description of these “Expedite fragments”, reference isagain made to WO 03/050531, as well as to the US provisional applicationof Ablynx N.V. entitled “Peptides capable of binding to serum proteins”of Ablynx N.V. (inventors: Revets, Hilde Adi Pierrette; Kolkman, JoostAlexander; and Hoogenboom, Hendricus Renerus Jacobus Mattheus) filed onDec. 5, 2006.

In one preferred, but non-limiting aspect, the invention relates to anamino acid sequence that binds to Tie2, blocks interaction with Ang1 andessentially consists of 4 framework regions and 3 complementaritydetermining regions, in which said amino acid sequence has at least 70%amino acid identity, preferably at least 80% amino acid identity, morepreferably at least 90% amino acid identity, such as 95% amino acididentity or more or less essentially 99 or 100% amino acid identity withthe sequence of at least one of the amino acid sequences of SEQ ID NO's:455 to 457, 459, or 460. This degree of amino acid identity can forexample be determined by determining the degree of amino acid identity(in a manner described herein) between said amino acid sequence and thesequences of SEQ ID NO's: 455 to 457, 459, or 460. Such amino acidsequences of the invention can be as further described herein, e.g.humanized and/or formatted into a multivalent and/or multispecificembodiment.

In a further preferred, but non-limiting aspect, the invention relatesto an amino acid sequence that binds to Ang2, blocks interaction withTie2 and essentially consists of 4 framework regions and 3complementarity determining regions, in which said amino acid sequencehas at least 70% amino acid identity, preferably at least 80% amino acididentity, more preferably at least 90% amino acid identity, such as 95%amino acid identity or more or less essentially 99 or 100% amino acididentity with the sequence of at least one of the amino acid sequencesof SEQ ID NO's: 464 to 469. This degree of amino acid identity can forexample be determined by determining the degree of amino acid identity(in a manner described herein) between said amino acid sequence and thesequences of SEQ ID NO's: 464 to 469. Such amino acid sequences of theinvention can be as further described herein, e.g. humanized and/orformatted into a multivalent and/or multispecific embodiment.

In another aspect, the invention relates to a compound or construct, andin particular a protein or polypeptide (also referred to herein as a“compound of the invention” or “polypeptide of the invention”,respectively) that comprises or essentially consists of one or moreamino acid sequences of the invention (or suitable fragments thereof),and optionally further comprises one or more other groups, residues,moieties or binding units. As will become clear to the skilled personfrom the further disclosure herein, such further groups, residues,moieties, binding units or amino acid sequences may or may not providefurther functionality to the amino acid sequence of the invention(and/or to the compound or construct in which it is present) and may ormay not modify the properties of the amino acid sequence of theinvention.

For example, such further groups, residues, moieties or binding unitsmay be one or more additional amino acid sequences, such that thecompound or construct is a (fusion) protein or (fusion) polypeptide. Ina preferred but non-limiting aspect, said one or more other groups,residues, moieties or binding units are immunoglobulin sequences. Evenmore preferably, said one or more other groups, residues, moieties orbinding units are chosen from the group consisting of domain antibodies,amino acid sequences that are suitable for use as a domain antibody,single domain antibodies, amino acid sequences that are suitable for useas a single domain antibody, “dAb”'s, amino acid sequences that aresuitable for use as a dAb, or Nanobodies.

Alternatively, such groups, residues, moieties or binding units may forexample be chemical groups, residues, moieties, which may or may not bythemselves be biologically and/or pharmacologically active. For example,and without limitation, such groups may be linked to the one or moreamino acid sequences of the invention so as to provide a “derivative” ofan amino acid sequence or polypeptide of the invention, as furtherdescribed herein.

Also within the scope of the present invention are compounds orconstructs, that comprises or essentially consists of one or morederivatives as described herein, and optionally further comprises one ormore other groups, residues, moieties or binding units, optionallylinked via one or more linkers. Preferably, said one or more othergroups, residues, moieties or binding units are amino acid sequences.

In the compounds or constructs described above, the one or more aminoacid sequences of the invention and the one or more groups, residues,moieties or binding units may be linked directly to each other and/orvia one or more suitable linkers or spacers. For example, when the oneor more groups, residues, moieties or binding units are amino acidsequences, the linkers may also be amino acid sequences, so that theresulting compound or construct is a fusion (protein) or fusion(polypeptide).

The compounds or polypeptides of the invention can generally be preparedby a method which comprises at least one step of suitably linking theone or more amino acid sequences of the invention to the one or morefurther groups, residues, moieties or binding units, optionally via theone or more suitable linkers, so as to provide the compound orpolypeptide of the invention. Polypeptides of the invention can also beprepared by a method which generally comprises at least the steps ofproviding a nucleic acid that encodes a polypeptide of the invention,expressing said nucleic acid in a suitable manner, and recovering theexpressed polypeptide of the invention. Such methods can be performed ina manner known per se, which will be clear to the skilled person, forexample on the basis of the methods and techniques further describedherein.

The process of designing/selecting and/or preparing a compound orpolypeptide of the invention, starting from an amino acid sequence ofthe invention, is also referred to herein as “formatting” said aminoacid sequence of the invention; and an amino acid of the invention thatis made part of a compound or polypeptide of the invention is said to be“formatted” or to be in the format of said compound or polypeptide ofthe invention. Examples of ways in which an amino acid sequence of theinvention can be formatted and examples of such formats will be clear tothe skilled person based on the disclosure herein; and such formattedamino acid sequences form a further aspect of the invention.

In one specific aspect of the invention, a compound of the invention ora polypeptide of the invention may have an increased half-life, comparedto the corresponding amino acid sequence of the invention. Somepreferred, but non-limiting examples of such compounds and polypeptideswill become clear to the skilled person based on the further disclosureherein, and for example comprise amino acid sequences or polypeptides ofthe invention that have been chemically modified to increase thehalf-life thereof (for example, by means of pegylation); amino acidsequences of the invention that comprise at least one additional bindingsite for binding to a serum protein (such as serum albumin); orpolypeptides of the invention that comprise at least one amino acidsequence of the invention that is linked to at least one moiety (and inparticular at least one amino acid sequence) that increases thehalf-life of the amino acid sequence of the invention. Examples ofpolypeptides of the invention that comprise such half-life extendingmoieties or amino acid sequences will become clear to the skilled personbased on the further disclosure herein; and for example include, withoutlimitation, polypeptides in which the one or more amino acid sequencesof the invention are suitable linked to one or more serum proteins orfragments thereof (such as (human) serum albumin or suitable fragmentsthereof) or to one or more binding units that can bind to serum proteins(such as, for example, domain antibodies, amino acid sequences that aresuitable for use as a domain antibody, single domain antibodies, aminoacid sequences that are suitable for use as a single domain antibody,“dAb”'s, amino acid sequences that are suitable for use as a dAb, orNanobodies that can bind to serum proteins such as serum albumin (suchas human serum albumin), serum immunoglobulins such as IgG, ortransferrine; reference is made to the further description andreferences mentioned herein); polypeptides in which an amino acidsequence of the invention is linked to an Fe portion (such as a humanFe) or a suitable part or fragment thereof; or polypeptides in which theone or more amino acid sequences of the invention are suitable linked toone or more small proteins or peptides that can bind to serum proteins(such as, without limitation, the proteins and peptides described in WO91/01743, WO 01/45746, WO 02/076489 and to the US provisionalapplication of Ablynx N.V. entitled “Peptides capable of binding toserum proteins” of Ablynx N.V. filed on Dec. 5, 2006.

Generally, the compounds or polypeptides of the invention with increasedhalf-life preferably have a half-life that is at least 1.5 times,preferably at least 2 times, such as at least 5 times, for example atleast 10 times or more than 20 times, greater than the half-life of thecorresponding amino acid sequence of the invention per se. For example,the compounds or polypeptides of the invention with increased half-lifemay have a half-life that is increased with more than 1 hours,preferably more than 2 hours, more preferably more than 6 hours, such asmore than 12 hours, or even more than 24, 48 or 72 hours, compared tothe corresponding amino acid sequence of the invention per se.

In a preferred, but non-limiting aspect of the invention, such compoundsor polypeptides of the invention have a serum half-life that isincreased with more than 1 hours, preferably more than 2 hours, morepreferably more than 6 hours, such as more than 12 hours, or even morethan 24.48 or 72 hours, compared to the corresponding amino acidsequence of the invention per se.

In another preferred, but non-limiting aspect of the invention, suchcompounds or polypeptides of the invention exhibit a serum half-life inhuman of at least about 12 hours, preferably at least 24 hours, morepreferably at least 48 hours, even more preferably at least 72 hours ormore. For example, compounds or polypeptides of the invention may have ahalf-life of at least 5 days (such as about 5 to 10 days), preferably atleast 9 days (such as about 9 to 14 days), more preferably at leastabout 10 days (such as about 10 to 15 days), or at least about 11 days(such as about 11 to 16 days), more preferably at least about 12 days(such as about 12 to 18 days or more), or more than 14 days (such asabout 14 to 19 days).

In another aspect, the invention relates to a nucleic acid that encodesan amino acid sequence of the invention or a polypeptide of theinvention (or a suitable fragment thereof). Such a nucleic acid willalso be referred to herein as a “nucleic acid of the invention” and mayfor example be in the form of a genetic construct, as further describedherein.

In another aspect, the invention relates to a host or host cell thatexpresses (or that under suitable circumstances is capable ofexpressing) an amino acid sequence of the invention and/or a polypeptideof the invention; and/or that contains a nucleic acid of the invention.Some preferred but non-limiting examples of such hosts or host cellswill become clear from the further description herein.

The invention further relates to a product or composition containing orcomprising at least one amino acid sequence of the invention, at leastone polypeptide of the invention (or a suitable fragment thereof) and/orat least one nucleic acid of the invention, and optionally one or morefurther components of such compositions known per se, i.e. depending onthe intended use of the composition. Such a product or composition mayfor example be a pharmaceutical composition (as described herein), aveterinary composition or a product or composition for diagnostic use(as also described herein). Some preferred but non-limiting examples ofsuch products or compositions will become clear from the furtherdescription herein.

The invention also relates to the use of an amino acid sequence,Nanobody or polypeptide of the invention, or of a composition comprisingthe same, in (methods or compositions for) modulating Tie1, Tie2, Ang1,Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6, either in vitro (e.g. in an in vitro or cellular assay) or invivo (e.g. in an a single cell or in a multicellular organism, and inparticular in a mammal, and more in particular in a human being, such asin a human being that is at risk of or suffers from a malignant,ischemic, inflammatory, infectious and immune disorder.

The invention also relates to methods for modulating Tie1, Tie2, Ang1Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6, either in vitro (e.g. in an in vitro or cellular assay) or invivo (e.g. in an a single cell or multicellular organism, and inparticular in a mammal, and more in particular in a human being, such asin a human being that is at risk of or suffers from a malignant,ischemic, inflammatory, infectious and immune disorder, which methodcomprises at least the step of contacting Tie1, Tie2, Ang1, Ang2, Ang3,Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6 with atleast one amino acid sequence, Nanobody or polypeptide of the invention,or with a composition comprising the same, in a manner and in an amountsuitable to modulate Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1,Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, with at least one aminoacid sequence, Nanobody or polypeptide of the invention.

The invention also relates to the use of an one amino acid sequence,Nanobody or polypeptide of the invention in the preparation of acomposition (such as, without limitation, a pharmaceutical compositionor preparation as further described herein) for modulating Tie1, Tie2,Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6, either in vitro (e.g. in an in vitro or cellular assay) or invivo (e.g. in an a single cell or multicellular organism, and inparticular in a mammal, and more in particular in a human being, such asin a human being that is at risk of or suffers from a malignant,ischemic, inflammatory, infectious and immune disorder.

In the context of the present invention, “modulating” or “to modulate”generally means either reducing or inhibiting the activity of, oralternatively increasing the activity of, Tie1, Tie2, Ang1, Ang2, Ang3,Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, asmeasured using a suitable in vitro, cellular or in vivo assay (such asthose mentioned herein). In particular, “modulating” or “to modulate”may mean either reducing or inhibiting the activity of, or alternativelyincreasing the activity of Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1,Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, as measured using asuitable in vitro, cellular or in vivo assay (such as those mentionedherein), by at least 1%, preferably at least 5%, such as at least 10% orat least 25%, for example by at least 50%, at least 60%, at least 70%,at least 80%, or 90% or more, compared to activity of Tie1, Tie2, Ang1,Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6 in the same assay under the same conditions but without thepresence of the amino acid sequence, Nanobody or polypeptide of theinvention.

As will be clear to the skilled person, “modulating” may also involveeffecting a change (which may either be an increase or a decrease) inaffinity, avidity, specificity and/or selectivity of Tie1, Tie2, Ang1,Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6 for one or more of its targets, ligands or substrates; and/oreffecting a change (which may either be an increase or a decrease) inthe sensitivity of Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,Angptl3, Angptl4, Angptl5, or Angptl6 for one or more conditions in themedium or surroundings in which Tie1, Tie2, Ang1, Ang2, Ang3, Ang4,Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6 is present (suchas pH, ion strength, the presence of co-factors, etc.), compared to thesame conditions but without the presence of the amino acid sequence,Nanobody or polypeptide of the invention. As will be clear to theskilled person, this may again be determined in any suitable mannerand/or using any suitable assay known per se, such as the assaysdescribed herein or in the prior art cited herein.

“Modulating” may also mean effecting a change (i.e. an activity as anagonist or as an antagonist, respectively) with respect to one or morebiological or physiological mechanisms, effects, responses, functions,pathways or activities in which Tie1, Tie2, Ang1, Ang2, Ang3, Ang4,Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6 is involved.Again, as will be clear to the skilled person, such an action as anagonist or an antagonist may be determined in any suitable manner and/orusing any suitable (in vitro and usually cellular or in assay) assayknown per se, such as the assays described herein or in the prior artcited herein. In particular, an action as an agonist or antagonist maybe such that an intended biological or physiological activity isincreased or decreased, respectively, by at least 1%, preferably atleast 5%, such as at least 10% or at least 25%, for example by at least50%, at least 60%, at least 70%, at least 80%, or 90% or more, comparedto the biological or physiological activity in the same assay under thesame conditions but without the presence of the amino acid sequence,Nanobody or polypeptide of the invention.

Modulating may for example involve reducing or inhibiting the binding ofTie1 or Tie2 to one of its substrates or ligands such as e.g. Ang1,Ang2, Ang3, Ang4. Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6 and/or competing with a natural ligand, substrate for binding.Modulating may also involve activating Tie1, Tie2, Ang1, Ang2, Ang3,Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6 or themechanism or pathway in which it is involved. Modulating may bereversible or irreversible, but for pharmaceutical and pharmacologicalpurposes will usually be in a reversible manner.

The invention further relates to methods for preparing or generating theamino acid sequences, polypeptides, nucleic acids, host cells, productsand compositions described herein. Some preferred but non-limitingexamples of such methods will become clear from the further descriptionherein.

Generally, these methods may comprise the steps of:

-   a) providing a set, collection or library of amino acid sequences;    and-   b) screening said set, collection or library of amino acid sequences    for amino acid sequences that can bind to and/or have affinity for    Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3,    Angptl4, Angptl5, or Angptl6;    and-   c) isolating the amino acid sequence(s) that can bind to and/or have    affinity for Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,    Angptl3, Angptl4, Angptl5, or Angptl6.

In such a method, the set, collection or library of amino acid sequencesmay be any suitable set, collection or library of amino acid sequences.For example, the set, collection or library of amino acid sequences maybe a set, collection or library of immunoglobulin sequences (asdescribed herein), such as a naïve set, collection or library ofimmunoglobulin sequences; a synthetic or semi-synthetic set, collectionor library of immunoglobulin sequences; and/or a set, collection orlibrary of immunoglobulin sequences that have been subjected to affinitymaturation.

Also, in such a method, the set, collection or library of amino acidsequences may be a set, collection or library of heavy chain variabledomains (such as V_(H) domains or V_(HH) domains) or of light chainvariable domains. For example, the set, collection or library of aminoacid sequences may be a set, collection or library of domain antibodiesor single domain antibodies, or may be a set, collection or library ofamino acid sequences that are capable of functioning as a domainantibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library ofamino acid sequences may be an immune set, collection or library ofimmunoglobulin sequences, for example derived from a mammal that hasbeen suitably immunized with Tie1, Tie2, Ang1, Ang2, Ang3, Ang4,Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6 or with asuitable antigenic determinant based thereon or derived there from, suchas an antigenic part, fragment, region, domain, loop or other epitopethereof. In one particular aspect, said antigenic determinant may be anextracellular part, region, domain, loop or other extracellularepitope(s).

In the above methods, the set, collection or library of amino acidsequences may be displayed on a phage, phagemid, ribosome or suitablemicro-organism (such as yeast), such as to facilitate screening.Suitable methods, techniques and host organisms for displaying andscreening (a set, collection or library of) amino acid sequences will beclear to the person skilled in the art, for example on the basis of thefurther disclosure herein. Reference is also made to the review byHoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

In another aspect, the method for generating amino acid sequencescomprises at least the steps of:

-   a) providing a collection or sample of cells expressing amino acid    sequences;-   b) screening said collection or sample of cells for cells that    express an amino acid sequence that can bind to and/or have affinity    for Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3,    Angptl4, Angptl5, or Angptl6;    and-   c) either (i) isolating said amino acid sequence; or (ii) isolating    from said cell a nucleic acid sequence that encodes said amino acid    sequence, followed by expressing said amino acid sequence.

For example, when the desired amino acid sequence is an immunoglobulinsequence, the collection or sample of cells may for example be acollection or sample of B-cells. Also, in this method, the sample ofcells may be derived from a mammal that has been suitably immunized withTie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4,Angptl5, or Angptl6 or with a suitable antigenic determinant basedthereon or derived there from, such as an antigenic part, fragment,region, domain, loop or other epitope thereof. In one particular aspect,said antigenic determinant may be an extracellular part, region, domain,loop or other extracellular epitope(s).

The above method may be performed in any suitable manner, as will beclear to the skilled person. Reference is for example made to EP 0 542810, WO 05/19824, WO 04/051268 and WO 04/106377. The screening of stepb) is preferably performed using a flow cytometry technique such asFACS. For this, reference is for example made to Lieby et al., Blood,Vol. 97, No. 12, 3820 (2001).

In another aspect, the method for generating an amino acid sequencedirected against Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,Angptl3, Angptl4, Angptl5, or Angptl6 may comprise at least the stepsof:

-   a) providing a set, collection or library of nucleic acid sequences    encoding amino acid sequences;-   b) screening said set, collection or library of nucleic acid    sequences for nucleic acid sequences that encode an amino acid    sequence that can bind to and/or has affinity for Tie1, Tie2, Ang1,    Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or    Angptl6;    and-   c) isolating said nucleic acid sequence, followed by expressing said    amino acid sequence.

In such a method, the set, collection or library of nucleic acidsequences encoding amino acid sequences may for example be a set,collection or library of nucleic acid sequences encoding a naïve set,collection or library of immunoglobulin sequences; a set, collection orlibrary of nucleic acid sequences encoding a synthetic or semi-syntheticset, collection or library of immunoglobulin sequences; and/or a set,collection or library of nucleic acid sequences encoding a set,collection or library of immunoglobulin sequences that have beensubjected to affinity maturation.

Also, in such a method, the set, collection or library of nucleic acidsequences may encode a set, collection or library of heavy chainvariable domains (such as V_(H) domains or V_(HH) domains) or of lightchain variable domains. For example, the set, collection or library ofnucleic acid sequences may encode a set, collection or library of domainantibodies or single domain antibodies, or a set, collection or libraryof amino acid sequences that are capable of functioning as a domainantibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library ofamino acid sequences may be an immune set, collection or library ofnucleic acid sequences, for example derived from a mammal that has beensuitably immunized with Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1,Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6 or with a suitableantigenic determinant based thereon or derived there from, such as anantigenic part, fragment, region, domain, loop or other epitope thereof.In one particular aspect, said antigenic determinant may be anextracellular part, region, domain, loop or other extracellularepitope(s).

The set, collection or library of nucleic acid sequences may for exampleencode an immune set, collection or library of heavy chain variabledomains or of light chain variable domains. In one specific aspect, theset, collection or library of nucleotide sequences may encode a set,collection or library of V_(HH) sequences.

In the above methods, the set, collection or library of nucleotidesequences may be displayed on a phage, phagemid, ribosome or suitablemicro-organism (such as yeast), such as to facilitate screening.Suitable methods, techniques and host organisms for displaying andscreening (a set, collection or library of) nucleotide sequencesencoding amino acid sequences will be clear to the person skilled in theart, for example on the basis of the further disclosure herein.Reference is also made to the review by Hoogenboom in NatureBiotechnology, 23, 9, 1105-1116 (2005).

The invention also relates to amino acid sequences that are obtained bythe above methods, or alternatively by a method that comprises the oneof the above methods and in addition at least the steps of determiningthe nucleotide sequence or amino acid sequence of said immunoglobulinsequence; and of expressing or synthesizing said amino acid sequence ina manner known per se, such as by expression in a suitable host cell orhost organism or by chemical synthesis.

Also, following the steps above, one or more amino acid sequences of theinvention may be suitably humanized (or alternatively camelized); and/orthe amino acid sequence(s) thus obtained may be linked to each other orto one or more other suitable amino acid sequences (optionally via oneor more suitable linkers) so as to provide a polypeptide of theinvention.

Also, a nucleic acid sequence encoding an amino acid sequence of theinvention may be suitably humanized (or alternatively camelized) andsuitably expressed; and/or one or more nucleic acid sequences encodingan amino acid sequence of the invention may be linked to each other orto one or more nucleic acid sequences that encode other suitable aminoacid sequences (optionally via nucleotide sequences that encode one ormore suitable linkers), after which the nucleotide sequence thusobtained may be suitably expressed so as to provide a polypeptide of theinvention.

The invention further relates to applications and uses of the amino acidsequences, compounds, constructs, polypeptides, nucleic acids, hostcells, products and compositions described herein, as well as to methodsfor the prevention and/or treatment for diseases and disordersassociated with Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,Angptl3, Angptl4, Angptl5, or Angptl6. Some preferred but non-limitingapplications and uses will become clear from the further descriptionherein.

The invention also relates to the amino acid sequences, compounds,constructs, polypeptides, nucleic acids, host cells, products andcompositions described herein for use in therapy.

In particular, the invention also relates to the amino acid sequences,compounds, constructs, polypeptides, nucleic acids, host cells, productsand compositions described herein for use in therapy of a disease ordisorder that can be prevented or treated by administering, to a subjectin need thereof, of (a pharmaceutically effective amount of) an aminoacid sequence, compound, construct or polypeptide as described herein.

More in particular, the invention relates to the amino acid sequences,compounds, constructs, polypeptides, nucleic acids, host cells, productsand compositions described herein for use in therapy of diseases anddisorders related to angiogenesis.

Other aspects, embodiments, advantages and applications of the inventionwill also become clear from the further description herein, in which theinvention will be described and discussed in more detail with referenceto the Nanobodies of the invention and polypeptides of the inventioncomprising the same, which form some of the preferred aspects of theinvention.

As will become clear from the further description herein, Nanobodiesgenerally offer certain advantages (outlined herein) compared to “dAb's”or similar (single) domain antibodies or immunoglobulin sequences, whichadvantages are also provided by the Nanobodies of the invention.However, it will be clear to the skilled person that the more generalaspects of the teaching below can also be applied (either directly oranalogously) to other amino acid sequences of the invention.

DETAILED DESCRIPTION OF THE INVENTION

In the present description, examples and claims:

-   a) Unless indicated or defined otherwise, all terms used have their    usual meaning in the art, which will be clear to the skilled person.    Reference is for example made to the standard handbooks, such as    Sambrook et al, “Molecular Cloning: A Laboratory Manual” (2″.Ed.),    Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et    al, eds., “Current protocols in molecular biology”, Green Publishing    and Wiley Interscience, New York (1987); Lewin, “Genes II”, John    Wiley & Sons, New York, N.Y., (1985); Old et al., “Principles of    Gene Manipulation: An Introduction to Genetic Engineering”, 2^(nd)    edition, University of California Press, Berkeley, Calif. (1981);    Roitt et al., “Immunology” (6^(th). Ed.), Mosby/Elsevier, Edinburgh    (2001); Roitt et al., Roitt's Essential Immunology, 10^(th) Ed.    Blackwell Publishing, UK (2001); and Janeway et al., “Immunobiology”    (6^(th) Ed.), Garland Science Publishing/Churchill Livingstone, New    York (2005), as well as to the general background art cited herein;-   b) Unless indicated otherwise, the term “immunoglobulin    sequence”—whether used herein to refer to a heavy chain antibody or    to a conventional 4-chain antibody—is used as a general term to    include both the full-size antibody, the individual chains thereof,    as well as all parts, domains or fragments thereof (including but    not limited to antigen-binding domains or fragments such as V_(HH)    ⁻¹ domains or V_(H)/V_(L), domains, respectively). In addition, the    term “sequence” as used herein (for example in terms like    “immunoglobulin sequence”, “antibody sequence”, “variable domain    sequence”, “V_(HH) sequence” or “protein sequence”), should    generally be understood to include both the relevant amino acid    sequence as well as nucleic acids or nucleotide sequences encoding    the same, unless the context requires a more limited interpretation.    Also, the term “nucleotide sequence” as used herein also encompasses    a nucleic acid molecule with said nucleotide sequence, so that the    terms “nucleotide sequence” and “nucleic acid” should be considered    equivalent and are used interchangeably herein;-   c) Unless indicated otherwise, all methods, steps, techniques and    manipulations that are not specifically described in detail can be    performed and have been performed in a manner known per se, as will    be clear to the skilled person. Reference is for example again made    to the standard handbooks and the general background art mentioned    herein and to the further references cited therein; as well as to    for example the following reviews Presta, Adv. Drug Deliv. Rev.    2006, 58 (5-6): 640-56; Levin and Weiss, Mol. Biosyst. 2006, 2(1):    49-57; Irving et al., J. Immunol. Methods, 2001, 248(1-2), 31-45;    Schmitz et al., Placenta, 2000, 21 Suppl. A, S106-12, Gonzales et    al., Tumour Biol., 2005, 26(1), 31-43, which describe techniques for    protein engineering, such as affinity maturation and other    techniques for improving the specificity and other desired    properties of proteins such as immunoglobulins.-   d) Amino acid residues will be indicated according to the standard    three-letter or one-letter amino acid code, as mentioned in Table    A-2;

TABLE A-2 one-letter and three-letter amino acid code Nonpolar, AlanineAla A uncharged Valine Val V (at pH 6.0-7.0)⁽³⁾ Leucine Leu L IsoleucineIle I Phenylalanine Phe F Methionine⁽¹⁾ Met M Tryptophan Trp W ProlinePro P Polar, Glycine⁽²⁾ Gly G uncharged Serine Ser S (at pH 6.0-7.0)Threonine Thr T Cysteine Cys C Asparagine Asn N Glutamine Gln Q TyrosineTyr Y Polar, Lysine Lys K charged Arginine Arg R (at pH 6.0-7.0)Histidine⁽⁴⁾ His H Aspartate Asp D Glutamate Glu E Notes: ⁽¹⁾Sometimesalso considered to be a polar uncharged amino acid. ⁽²⁾Sometimes alsoconsidered to be a nonpolar uncharged amino acid. ⁽³⁾As will be clear tothe skilled person, the fact that an amino acid residue is referred toin this Table as being either charged or uncharged at pH 6.0 to 7.0 doesnot reflect in any way on the charge said amino acid residue may have ata pH lower than 6.0 and/or at a pH higher than 7.0; the amino acidresidues mentioned in the Table can be either charged and/or unchargedat such a higher or lower pH, as will be clear to the skilled person.⁽⁴⁾As is known in the art, the charge of a His residue is greatlydependant upon even small shifts in pH, but a His residu can generallybe considered essentially uncharged at a pH of about 6.5.

-   e) For the purposes of comparing two or more nucleotide sequences,    the percentage of “sequence identity” between a first nucleotide    sequence and a second nucleotide sequence may be calculated by    dividing [the number of nucleotides in the first nucleotide sequence    that are identical to the nucleotides at the corresponding positions    in the second nucleotide sequence] by [the total number of    nucleotides in the first nucleotide sequence] and multiplying by    [100%], in which each deletion, insertion, substitution or addition    of a nucleotide in the second nucleotide sequence—compared to the    first nucleotide sequence—is considered as a difference at a single    nucleotide (position).    -   Alternatively, the degree of sequence identity between two or        more nucleotide sequences may be calculated using a known        computer algorithm for sequence alignment such as NCBI Blast        v2.0, using standard settings.    -   Some other techniques, computer algorithms and settings for        determining the degree of sequence identity are for example        described in WO 04/037999, EP 0 967 284, EP 1 085 089, WO        00/55318, WO 00/78972, WO 98/49185 and GB 2 357 768-A.    -   Usually, for the purpose of determining the percentage of        “sequence identity” between two nucleotide sequences in        accordance with the calculation method outlined hereinabove, the        nucleotide sequence with the greatest number of nucleotides will        be taken as the “first” nucleotide sequence, and the other        nucleotide sequence will be taken as the “second” nucleotide        sequence;-   f) For the purposes of comparing two or more amino acid sequences,    the percentage of “sequence identity” between a first amino acid    sequence and a second amino acid sequence (also referred to herein    as “amino acid identity”) may be calculated by dividing [the number    of amino acid residues in the first amino acid sequence that are    identical to the amino acid residues at the corresponding positions    in the second amino acid sequence] by [the total number of amino    acid residues in the first amino acid sequence] and multiplying by    [100%], in which each deletion, insertion, substitution or addition    of an amino acid residue in the second amino acid sequence—compared    to the first amino acid sequence—is considered as a difference at a    single amino acid residue (position), i.e. as an “amino acid    difference” as defined herein.    -   Alternatively, the degree of sequence identity between two amino        acid sequences may be calculated using a known computer        algorithm, such as those mentioned above for determining the        degree of sequence identity for nucleotide sequences, again        using standard settings.    -   Usually, for the purpose of determining the percentage of        “sequence identity” between two amino acid sequences in        accordance with the calculation method outlined hereinabove, the        amino acid sequence with the greatest number of amino acid        residues will be taken as the “first” amino acid sequence, and        the other amino acid sequence will be taken as the “second”        amino acid sequence.    -   Also, in determining the degree of sequence identity between two        amino acid sequences, the skilled person may take into account        so-called “conservative” amino acid substitutions, which can        generally be described as amino acid substitutions in which an        amino acid residue is replaced with another amino acid residue        of similar chemical structure and which has little or        essentially no influence on the function, activity or other        biological properties of the polypeptide. Such conservative        amino acid substitutions are well known in the art, for example        from WO 04/037999, GB-A-3 357 768, WO 98/49185, WO 00/46383 and        WO 01/09300; and (preferred) types and/or combinations of such        substitutions may be selected on the basis of the pertinent        teachings from WO 04/037999 as well as WO 98/49185 and from the        further references cited therein.    -   Such conservative substitutions preferably are substitutions in        which one amino acid within the following groups (a)-(e) is        substituted by another amino acid residue within the same        group: (a) small aliphatic, nonpolar or slightly polar residues:        Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged        residues and their (uncharged) amides: Asp, Asn, Glu and        Gln; (c) polar, positively charged residues: His, Arg and        Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val        and Cys; and (e) aromatic residues: Phe, Tyr and Trp.    -   Particularly preferred conservative substitutions are as        follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gln or        into His; Asp into Glu; Cys into Ser; Gln into Asn; Glu into        Asp; Gly into Ala or into Pro; His into Asn or into Gln; Ile        into Len or into Val; Leu into Ile or into Val; Lys into Arg,        into Gln or into Glu; Met into Leu, into Tyr or into Ile; Phe        into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp        into Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into        Leu.    -   Any amino acid substitutions applied to the polypeptides        described herein may also be based on the analysis of the        frequencies of amino acid variations between homologous proteins        of different species developed by Schulz et al., Principles of        Protein Structure, Springer-Verlag, 1978, on the analyses of        structure forming potentials developed by Chou and Fasman,        Biochemistry 13: 211, 1974 and Adv. Enzymol., 47: 45-149, 1978,        and on the analysis of hydrophobicity patterns in proteins        developed by Eisenberg et al., Proc. Nad. Acad. Sci. USA 81:        140-144, 1984; Kyte & Doolittle; J. Molec. Biol. 157: 105-132,        198 I, and Goldman et al., Aim. Rev. Biophys. Chem. 15: 321-353,        1986, all incorporated herein in their entirety by reference.        Information on the primary, secondary and tertiary structure of        Nanobodies is given in the description herein and in the general        background art cited above. Also, for this purpose, the crystal        structure of a V_(HH) domain from a llama is for example given        by Desmyter et al., Nature Structural Biology, Vol. 3, 9, 803        (1996); Spinelli et al., Natural Structural Biology (1996); 3,        752-757; and Decanniere et al., Structure, Vol. 7, 4, 361        (1999). Further information about some of the amino acid        residues that in conventional V_(H) domains form the V_(H)/V_(L)        interface and potential camelizing substitutions on these        positions can be found in the prior art cited above.-   g) Amino acid sequences and nucleic acid sequences are said to be    “exactly the same” if they have 100% sequence identity (as defined    herein) over their entire length;-   h) When comparing two amino acid sequences, the term “amino acid    difference” refers to an insertion, deletion or substitution of a    single amino acid residue on a position of the first sequence,    compared to the second sequence; it being understood that two amino    acid sequences can contain one, two or more such amino acid    differences;-   i) When a nucleotide sequence or amino acid sequence is said to    “comprise” another nucleotide sequence or amino acid sequence,    respectively, or to “essentially consist of” another nucleotide    sequence or amino acid sequence, this may mean that the latter    nucleotide sequence or amino acid sequence has been incorporated    into the first mentioned nucleotide sequence or amino acid sequence,    respectively, but more usually this generally means that the first    mentioned nucleotide sequence or amino acid sequence comprises    within its sequence a stretch of nucleotides or amino acid residues,    respectively, that has the same nucleotide sequence or amino acid    sequence, respectively, as the latter sequence, irrespective of how    the first mentioned sequence has actually been generated or obtained    (which may for example be by any suitable method described herein).    By means of a non-limiting example, when a Nanobody of the invention    is said to comprise a CDR sequence, this may mean that said CDR    sequence has been incorporated into the Nanobody of the invention,    but more usually this generally means that the Nanobody of the    invention contains within its sequence a stretch of amino acid    residues with the same amino acid sequence as said CDR sequence,    irrespective of how said Nanobody of the invention has been    generated or obtained. It should also be noted that when the latter    amino acid sequence has a specific biological or structural    function, it preferably has essentially the same, a similar or an    equivalent biological or structural function in the first mentioned    amino acid sequence (in other words, the first mentioned amino acid    sequence is preferably such that the latter sequence is capable of    performing essentially the same, a similar or an equivalent    biological or structural function). For example, when a Nanobody of    the invention is said to comprise a CDR sequence or framework    sequence, respectively, the CDR sequence and framework are    preferably capable, in said Nanobody, of functioning as a CDR    sequence or framework sequence, respectively. Also, when a    nucleotide sequence is said to comprise another nucleotide sequence,    the first mentioned nucleotide sequence is preferably such that,    when it is expressed into an expression product (e.g. a    polypeptide), the amino acid sequence encoded by the latter    nucleotide sequence forms part of said expression product (in other    words, that the latter nucleotide sequence is in the same reading    frame as the first mentioned, larger nucleotide sequence).-   j) A nucleic acid sequence or amino acid sequence is considered to    be “(in) essentially isolated (form)”—for example, compared to its    native biological source and/or the reaction medium or cultivation    medium from which it has been obtained—when it has been separated    from at least one other component with which it is usually    associated in said source or medium, such as another nucleic acid,    another protein/polypeptide, another biological component or    macromolecule or at least one contaminant, impurity or minor    component. In particular, a nucleic acid sequence or amino acid    sequence is considered “essentially isolated” when it has been    purified at least 2-fold, in particular at least 10-fold, more in    particular at least 100-fold, and up to 1000-fold or more. A nucleic    acid sequence or amino acid sequence that is “in essentially    isolated form” is preferably essentially homogeneous, as determined    using a suitable technique, such as a suitable chromatographical    technique, such as polyacrylamide-gel electrophoresis;-   k) The term “domain” as used herein generally refers to a globular    region of an amino acid sequence (such as an antibody chain, and in    particular to a globular region of a heavy chain antibody), or to a    polypeptide that essentially consists of such a globular region.    Usually, such a domain will comprise peptide loops (for example 3 or    4 peptide loops) stabilized, for example, as a sheet or by disulfide    bonds. The term “binding domain” refers to such a domain that is    directed against an antigenic determinant (as defined herein);-   l) The term “antigenic determinant” refers to the epitope on the    antigen recognized by the antigen-binding molecule (such as a    Nanobody or a polypeptide of the invention) and more in particular    by the antigen-binding site of said molecule. The terms “antigenic    determinant” and “epitope” may also be used interchangeably herein.-   m) An amino acid sequence (such as a Nanobody, an antibody, a    polypeptide of the invention, or generally an antigen binding    protein or polypeptide or a fragment thereof) that can    (specifically) bind to, that has affinity for and/or that has    specificity for a specific antigenic determinant, epitope, antigen    or protein (or for at least one part, fragment or epitope thereof)    is said to be “against” or “directed against” said antigenic    determinant, epitope, antigen or protein.-   n) The term “specificity” refers to the number of different types of    antigens or antigenic determinants to which a particular    antigen-binding molecule or antigen-binding protein (such as a    Nanobody or a polypeptide of the invention) molecule can bind. The    specificity of an antigen-binding protein can be determined based on    affinity and/or avidity. The affinity, represented by the    equilibrium constant for the dissociation of an antigen with an    antigen-binding protein (K_(D)), is a measure for the binding    strength between an antigenic determinant and an antigen-binding    site on the antigen-binding protein: the lesser the value of the    K_(D), the stronger the binding strength between an antigenic    determinant and the antigen-binding molecule (alternatively, the    affinity can also be expressed as the affinity constant (K_(A)),    which is 1/K_(D)). As will be clear to the skilled person (for    example on the basis of the further disclosure herein), affinity can    be determined in a manner known per se, depending on the specific    antigen of interest. Avidity is the measure of the strength of    binding between an antigen-binding molecule (such as a Nanobody or    polypeptide of the invention) and the pertinent antigen. Avidity is    related to both the affinity between an antigenic determinant and    its antigen binding site on the antigen-binding molecule and the    number of pertinent binding sites present on the antigen-binding    molecule. Typically, antigen-binding proteins (such as the amino    acid sequences, Nanobodies and/or polypeptides of the invention)    will bind to their antigen with a dissociation constant (K_(D)) of    10⁻⁵ to 10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²    moles/liter or less and more preferably 10⁻⁸ to 10⁻¹² moles/liter    (i.e. with an association constant (K_(A)) of 10⁵ to 10¹²    liter/moles or more, and preferably 10⁷ to 10¹² liter/moles or more    and more preferably 10⁸ to 10¹² liter/moles). Any K_(D) value    greater than 10⁴ mol/liter (or any K_(A) value lower than 10⁴ M⁻¹)    liters/mol is generally considered to indicate non-specific binding.    Preferably, a monovalent immunoglobulin sequence of the invention    will bind to the desired antigen with an affinity less than 500 nM,    preferably less than 200 nM, more preferably less than 10 nM, such    as less than 500 pM. Specific binding of an antigen-binding protein    to an antigen or antigenic determinant can be determined in any    suitable manner known per se, including, for example, Scatchard    analysis and/or competitive binding assays, such as radio    immunoassay (RIA), enzyme immunoassays (EIA) and sandwich    competition assays, and the different variants thereof known per se    in the art; as well as the other techniques mentioned herein.    -   The dissociation constant may be the actual or apparent        dissociation constant, as will be clear to the skilled person.        Methods for determining the dissociation constant will be clear        to the skilled person, and for example include the techniques        mentioned herein. In this respect, it will also be clear that it        may not be possible to measure dissociation constants of more        then 10⁻¹ moles/liter or 10⁻³ moles/liter (e.g., of 10⁻²        moles/liter). Optionally, as will also be clear to the skilled        person, the (actual or apparent) dissociation constant may be        calculated on the basis of the (actual or apparent) association        constant (K_(A)), by means of the relationship [K_(D)=1/K_(A)].    -   The affinity denotes the strength or stability of a molecular        interaction. The affinity is commonly given as by the K_(D), or        dissociation constant, which has units of mol/liter (or M). The        affinity can also be expressed as an association constant,        K_(A), which equals 1/K_(D) and has units of (mol/liter)⁻¹ (or        M⁻¹). In the present specification, the stability of the        interaction between two molecules (such as an amino acid        sequence, Nanobody or polypeptide of the invention and its        intended target) will mainly be expressed in terms of the K_(D)        value of their interaction; it being clear to the skilled person        that in view of the relation K_(A)=1/K_(D), specifying the        strength of molecular interaction by its K_(D) value can also be        used to calculate the corresponding K_(A) value. The K_(D)-value        characterizes the strength of a molecular interaction also in a        thermodynamic sense as it is related to the free energy (DG) of        binding by the well known relation. DG=RT. ln(K_(D))        (equivalently DG=−RT. ln(K_(A))), where R equals the gas        constant, T equals the absolute temperature and in denotes the        natural logarithm.    -   The K_(D) for biological interactions which are considered        meaningful (e.g. specific) are typically in the range of 10⁻¹⁰ M        (0.1 nM) to 10⁻⁵ M (10000 nM). The stronger an interaction is        the lower is its K_(D).    -   The K_(D) can also be expressed as the ratio of the dissociation        rate constant of a complex, denoted as k_(off), to the rate of        its association, denoted k_(on) (so that K_(D)=k_(off)/k_(on)        and K_(A)=k_(on)/k_(off)). The off-rate k_(off) has unit's s⁻¹        (where s is the SI unit notation of second). The on-rate k_(on)        has units M⁻¹s⁻¹. The on-rate may vary between 10² M⁻¹s⁻¹ to        about 10⁷ M⁻¹s⁻¹, approaching the diffusion-limited association        rate constant for bimolecular interactions. The off-rate is        related to the half-life of a given molecular interaction by the        relation t_(1/2)=ln (2)/k_(off). The off-rate may vary between        10⁻⁶ s⁻¹ (near irreversible complex with a t_(1/2) of multiple        days) to 1 s⁻¹ (t_(1/2)-0.69 s).    -   The affinity of a molecular interaction between two molecules        can be measured via different techniques known per se, such as        the well known surface plasmon resonance (SPR) biosensor        technique (see for example Ober et al., Intern. Immunology, 13,        1551-1559, 2001) where one molecule is immobilized on the        biosensor chip and the other molecule is passed over the        immobilized molecule under flow conditions yielding k_(on),        k_(off) measurements and hence K_(D) (or K_(A)) values. This can        for example be performed using the well-known BIACORE        instruments.    -   It will also be clear to the skilled person that the measured        K_(D) may correspond to the apparent K_(D) if the measuring        process somehow influences the intrinsic binding affinity of the        implied molecules for example by artefacts related to the        coating on the biosensor of one molecule. Also, an apparent        K_(D) may be measured if one molecule contains more than one        recognition sites for the other molecule. In such situation the        measured affinity may be affected by the avidity of the        interaction by the two molecules.    -   Another approach that may be used to assess affinity is the        2-step ELISA (Enzyme-Linked Immunosorbent Assay) procedure of        Friguet et al. (J. Immunol. Methods, 77, 305-19, 1985). This        method establishes a solution phase binding equilibrium        measurement and avoids possible artefacts relating to adsorption        of one of the molecules on a support such as plastic.    -   However, the accurate measurement of K_(D) may be quite        labour-intensive and as consequence, often apparent K_(D) values        are determined to assess the binding strength of two molecules.        It should be noted that as long all measurements are made in a        consistent way (e.g. keeping the assay conditions unchanged)        apparent K_(D) measurements can be used as an approximation of        the true K_(D) and hence in the present document K_(D) and        apparent K_(D) should be treated with equal importance or        relevance. Finally, it should be noted that in many situations        the experienced scientist may judge it to be convenient to        determine the binding affinity relative to some reference        molecule. For example, to assess the binding strength between        molecules A and B, one may e.g. use a reference molecule C that        is known to bind to B and that is suitably labelled with a        fluorophore or chromophore group or other chemical moiety, such        as biotin for easy detection in an ELISA or FACS (Fluorescent        activated cell sorting) or other format (the fluorophore for        fluorescence detection, the chromophore for light absorption        detection, the biotin for streptavidin-mediated ELISA        detection.). Typically, the reference molecule C is kept at a        fixed concentration and the concentration of A is varied for a        given concentration or amount of B. As a result an IC₅₀ value is        obtained corresponding to the concentration of A at which the        signal measured for C in absence of A is halved. Provided        K_(D ref), the K_(D) of the reference molecule, is known, as        well as the total concentration c_(ref) of the reference        molecule, the apparent K_(D) for the interaction A-B can be        obtained from following formula:        K_(D)=IC₅₀/(1+c_(ref)/K_(D ref)). Note that if        c_(ref)<<K_(D ref), K_(D)≈IC₅₀. Provided the measurement of the        IC₅₀ is performed in a consistent way (e.g. keeping c_(ref)        fixed) for the binders that are compared, the strength or        stability of a molecular interaction can be assessed by the IC₅₀        and this measurement is judged as equivalent to K_(D) or to        apparent K_(D) throughout this text.-   o) The half-life of an amino acid sequence, compound or polypeptide    of the invention can generally be defined as the time taken for the    serum concentration of the amino acid sequence, compound or    polypeptide to be reduced by 50%, in vivo, for example due to    degradation of the sequence or compound and/or clearance or    sequestration of the sequence or compound by natural mechanisms. The    in vivo half-life of an amino acid sequence, compound or polypeptide    of the invention can be determined in any manner known per se, such    as by pharmacokinetic analysis. Suitable techniques will be clear to    the person skilled in the art, and may for example generally involve    the steps of suitably administering to a warm-blooded animal (i.e.    to a human or to another suitable mammal, such as a mouse, rabbit,    rat, pig, dog or a primate, for example monkeys from the genus    Macaca (such as, and in particular, cynomologus monkeys (Macaca    fascicularis) and/or rhesus monkeys (Macaca mulatta)) and baboon    (Papio ursinus)) a suitable dose of the amino acid sequence,    compound or polypeptide of the invention; collecting blood samples    or other samples from said animal; determining the level or    concentration of the amino acid sequence, compound or polypeptide of    the invention in said blood sample; and calculating, from (a plot    of) the data thus obtained, the time until the level or    concentration of the amino acid sequence, compound or polypeptide of    the invention has been reduced by 50% compared to the initial level    upon dosing. Reference is for example made to the Experimental Part    below, as well as to Dennis et al., J. Biol. Chem. 277:35035-42    (2002), and to the standard handbooks, such as Kenneth, A et al:    Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists    and Peters et al, Pharmacokinete analysis: A Practical Approach    (1996). Reference is also made to “Pharmacokinetics”, M Gibaldi & D    Perron, published by Marcel Dekker, 2nd Rev. edition (1982).    -   As will also be clear to the skilled person (see for example        pages 6 and 7 of WO 04/003019 and in the further references        cited therein), the half-life can be expressed using parameters        such as the t1/2-alpha, t1/2-beta and the area under the curve        (AUC). In the present specification, an “increase in half-life”        refers to an increase in any one of these parameters, such as        any two of these parameters, or essentially all three these        parameters. As used herein “increase in half-life” or “increased        half-life” in particular refers to an increase in the t1/2-beta,        either with or without an increase in the t1/2-alpha and/or the        AUC or both.    -   For example, the half-life of an amino acid sequence or        polypeptide of the invention may be determined by means of a        pharmacokinetic study, performed in a rodent or non-human        primate model, as follows. Groups of animals (n=2-10) are given        an intravenous bolus injection of 1 mg/kg or 10 mg/kg 2D3-17D12        fusion protein. Plasma samples are obtained via a vein at        different timepoints after dosing (eg. 1, 2, 4, 6, 8, 12, 24,        48, 144, 192, 240, 288 and 336 h after dosing) and analyzed for        the presence of the 2D3-17D12 fusion protein by ELISA. Plasma        concentration versus time are fitted to a two-compartment        elimination model. The pharmacokinetic parameters of clearance,        V1, steady state volume (Vss), T½, AUC, and AUC corrected for        actual dose administered (AUC/dose) are averaged for each        treatment group. Differences between groups are determined by        analysis of variance.-   p) In the context of the present invention, “modulating” or “to    modulate” generally means either reducing or inhibiting the activity    of, or alternatively increasing the activity of, a target or    antigen, as measured using a suitable in vitro, cellular or in vivo    assay. In particular, “modulating” or “to modulate” may mean either    reducing or inhibiting the activity of, or alternatively increasing    a (relevant or intended) biological activity of, a target or    antigen, as measured using a suitable in vitro, cellular or in vivo    assay (which will usually depend on the target or antigen involved),    by at least 1%, preferably at least 5%, such as at least 10% or at    least 25%, for example by at least 50%, at least 60%, at least 70%,    at least 80%, or 90% or more, compared to activity of the target or    antigen in the same assay under the same conditions but without the    presence of the construct of the invention.    -   As will be clear to the skilled person, “modulating” may also        involve effecting a change (which may either be an increase or a        decrease) in affinity, avidity, specificity and/or selectivity        of a target or antigen for one or more of its ligands, binding        partners, partners for association into a homomultimeric or        heteromultimeric form, or substrates; and/or effecting a change        (which may either be an increase or a decrease) in the        sensitivity of the target or antigen for one or more conditions        in the medium or surroundings in which the target or antigen is        present (such as pH, ion strength, the presence of co-factors,        etc.), compared to the same conditions but without the presence        of the construct of the invention. As will be clear to the        skilled person, this may again be determined in any suitable        manner and/or using any suitable assay known per se, depending        on the target or antigen involved.    -   “Modulating” may also mean effecting a change (i.e. an activity        as an agonist, as an antagonist or as a reverse agonist,        respectively, depending on the target or antigen and the desired        biological or physiological effect) with respect to one or more        biological or physiological mechanisms, effects, responses,        functions, pathways or activities in which the target or antigen        (or in which its substrate(s), ligand(s) or pathway(s) are        involved, such as its signalling pathway or metabolic pathway        and their associated biological or physiological effects) is        involved. Again, as will be clear to the skilled person, such an        action as an agonist or an antagonist may be determined in any        suitable manner and/or using any suitable (in vitro and usually        cellular or in assay) assay known per se, depending on the        target or antigen involved. In particular, an action as an        agonist or antagonist may be such that an intended biological or        physiological activity is increased or decreased, respectively,        by at least 1%, preferably at least 5%, such as at least 10% or        at least 25%, for example by at least 50%, at least 60%, at        least 70%, at least 80%, or 90% or more, compared to the        biological or physiological activity in the same assay under the        same conditions but without the presence of the construct of the        invention.    -   Modulating may for example also involve allosteric modulation of        the target or antigen; and/or reducing or inhibiting the binding        of the target or antigen to one of its substrates or ligands        and/or competing with a natural ligand, substrate for binding to        the target or antigen. Modulating may also involve activating        the target or antigen or the mechanism or pathway in which it is        involved. Modulating may for example also involve effecting a        change in respect of the folding or confirmation of the target        or antigen, or in respect of the ability of the target or        antigen to fold, to change its confirmation (for example, upon        binding of a ligand), to associate with other (sub)units, or to        disassociate. Modulating may for example also involve effecting        a change in the ability of the target or antigen to transport        other compounds or to serve as a channel for other compounds        (such as ions).    -   Modulating may be reversible or irreversible, but for        pharmaceutical and pharmacological purposes will usually be in a        reversible manner,-   o) The invention also provides amino acid sequences that cross-block    the binding of one of the amino acid sequences described in this    application and/or are cross-blocked from binding Ang, Angptl, or    Tie by one of the amino acid sequences described in this    application. The terms “cross-block”, “cross-blocked” and    “cross-blocking” are used interchangeably herein to mean the ability    of an amino acid sequence or other binding agents to interfere with    the binding of other amino acid sequences or binding agents of the    invention to Ang, Angptl, or Tie. The extend to which an amino acid    sequence or other binding agents of the invention is able to    interfere with the binding of another to Ang, Angptl, or Tie, and    therefore whether it can be said to cross-block according to the    invention, can be determined using competition binding assays. One    particularly suitable quantitative assay uses a Biacore machine    which can measure the extent of interactions using surface plasmon    resonance technology. Another suitable quantitative cross-blocking    assay uses an ELISA-based approach to measure competition between    amino acid sequence or another binding agents in terms of their    binding to Ang, Angptl, or Tie. Other preferred amino acid sequences    of the invention are amino acid sequences comprising at least one    single variable domain that cross-block at least one amino acid    sequence with SEQ ID NOs 455 to 501 or are cross-blocked by the at    least one amino acid sequence with SEQ ID NOs 455 to 501. The    following generally describes a suitable Biacore assay for    determining whether an amino acid sequence or other binding agent    cross-blocks or is capable of cross-blocking according to the    invention. It will be appreciated that the assay can be used with    any of the Ang, Angptl, or Tie binding agents described herein. The    Biacore machine (for example the Biacore 3000) is operated in line    with the manufacturer's recommendations. Thus in one cross-blocking    assay, the Ang, Angptl, or Tie protein is coupled to a CM5 Biacore    chip using standard amine coupling chemistry to generate a Ang,    Angptl, or Tie-coated surface. Typically 200-800 resonance units of    Ang, Angptl, or Tie would be coupled to the chip (an amount that    gives easily measurable levels of binding but that is readily    saturable by the concentrations of test reagent being used). Two    test amino acid sequences (termed A* and B*) to be assessed for    their ability to cross-block each other are mixed at a one to one    molar ratio of binding sites in a suitable buffer to create the test    mixture. When calculating the concentrations on a binding site basis    the molecular weight of an amino acid sequence is assumed to be the    total molecular weight of the amino acid sequence divided by the    number of Ang, Angptl, or Tie binding sites on that amino acid    sequence. The concentration of each amino acid sequence in the test    mix should be high enough to readily saturate the binding sites for    that amino acid sequence on the Ang, Angptl, or Tie molecules    captured on the Biacore chip. The amino acid sequences in the    mixture are at the same molar concentration (on a binding basis) and    that concentration would typically be between 1.00 and 1.5    micromolar (on a binding site basis). Separate solutions containing    A* alone and B* alone are also prepared. A* and B* in these    solutions should be in the same buffer and at the same concentration    as in the test mix. The test mixture is passed over the Ang, Angptl,    or Tie-coated Biacore chip and the total amount of binding recorded.    The chip is then treated in such a way as to remove the bound amino    acid sequences without damaging the chip-bound Ang, Angptl, or Tie.    Typically this is done by treating the chip with 30 mM HCl for 60    seconds. The solution of A* alone is then passed over the Ang,    Angptl, or Tie-coated surface and the amount of binding recorded.    The chip is again treated to remove all of the bound amino acid    sequences without damaging the chip-bound Ang, Angptl, or Tie. The    solution of B* alone is then passed over the Ang, Angptl, or    Tie-coated surface and the amount of binding recorded. The maximum    theoretical binding of the mixture of A* and B* is next calculated,    and is the sum of the binding of each amino acid sequence when    passed over the Ang, Angptl, or Tie surface alone. If the actual    recorded binding of the mixture is less than this theoretical    maximum then the two amino acid sequences are cross-blocking each    other. Thus, in general, a cross-blocking amino acid sequence or    other binding agent according to the invention is one which will    bind to Ang, Angptl, or Tie in the above Biacore cross-blocking    assay such that during the assay and in the presence of a second    amino acid sequence or other binding agent of the invention the    recorded binding is between 80% and 0.1% (e.g. 80% to 4%) of the    maximum theoretical binding, specifically between 75% and 0.1% (e.g.    75% to 4%) of the maximum theoretical binding, and more specifically    between 70% and 0.1% (e.g. 70% to 4%) of maximum theoretical binding    (as just defined above) of the two amino acid sequences or binding    agents in combination. The Biacore assay described above is a    primary assay used to determine if amino acid sequences or other    binding agents cross-block each other according to the invention. On    rare occasions particular amino acid sequences or other binding    agents may not bind to Ang, Angptl, or Tie coupled via amine    chemistry to a CM5 Biacore chip (this usually occurs when the    relevant binding site on Ang, Angptl, or Tie is masked or destroyed    by the coupling to the chip). In such eases cross-blocking can be    determined using a tagged version of Ang, Angptl, or Tie, for    example N-terminal His-tagged Ang, Angptl, or Tie (R & D Systems,    Minneapolis, Minn., USA; 2005 cat#1406-ST-025). In this particular    format, an anti-His amino acid sequence would be coupled to the    Biacore chip and then the His-tagged Ang, Angptl, or Tie would be    passed over the surface of the chip and captured by the anti-His    amino acid sequence. The cross blocking analysis would be carried    out essentially as described above, except that after each chip    regeneration cycle, new His-tagged Ang, Angptl, or Tie would be    loaded back onto the anti-His amino acid sequence coated surface. In    addition to the example given using N-terminal His-tagged Ang,    Angptl, or Tie, C-terminal His-tagged Ang, Angptl, or Tie could    alternatively be used. Furthermore, various other tags and tag    binding protein combinations that are known in the art could be used    for such a cross-blocking analysis (e.g. HA tag with anti-HA    antibodies; FLAG tag with anti-FLAG antibodies; biotin tag with    streptavidin). The following generally describes an ELISA assay for    determining whether an anti-Ang. Angptl, or Tie amino acid sequence    or other Ang, Angptl, or Tie binding agent cross-blocks or is    capable of cross-blocking according to the invention. It will be    appreciated that the assay can be used with any of the Ang, Angptl,    or Tie binding agents described herein. The general principal of the    assay is to have an anti-Ang, Angptl, or Tie amino acid sequence    coated onto the wells of an ELISA plate. An excess amount of a    second, potentially cross-blocking, anti-Ang, Angptl, or Tie amino    acid sequence is added in solution (i.e. not bound to the ELISA    plate). A limited amount of Ang, Angptl, or Tie is then added to the    wells. The coated amino acid sequence and the amino acid sequence in    solution compete for binding of the limited number of Ang, Angptl,    or Tie molecules. The plate is washed to remove Ang, Angptl, or Tie    that has not been bound by the coated [amino acid sequence] and to    also remove the second, solution phase amino acid sequence as well    as any complexes formed between the second, solution phase amino    acid sequence and Ang, Angptl, or Tie. The amount of bound Ang,    Angptl, or Tie is then measured using an appropriate Ang, Angptl, or    Tie detection reagent. An amino acid sequence in solution that is    able to cross-block the coated amino acid sequence will be able to    cause a decrease in the number of Ang, Angptl, or Tie molecules that    the coated amino acid sequence can bind relative to the number of    Ang, Angptl, or Tie molecules that the coated amino acid sequence    can bind in the absence of the second, solution phase, amino acid    sequence. In the instance where the first amino acid sequence, e.g.    an Nanobody-X, is chosen to be the immobilized amino acid sequence,    it is coated onto the wells of the ELISA plate, after which the    plates are blocked with a suitable blocking solution to minimize    non-specific binding of reagents that are subsequently added. An    excess amount of the second amino acid sequence, i.e. Nanobody-Y, is    then added to the ELISA plate such that the moles of Nanobody-Y Ang,    Angptl, or Tie binding sites per well are at least 10 fold higher    than the moles of Nanobody-X Ang, Angptl, or Tie binding sites that    were used, per well, during the coating of the ELISA plate. Ang.    Angptl, or Tie is then added such that the moles of Ang, Angptl, or    Tie added per well are at least 25-fold lower than the moles of    Nanobody-X Ang, Angptl, or Tie binding sites that were used for    coating each well. Following a suitable incubation period the ELISA    plate is washed and a Ang, Angptl, or Tie detection reagent is added    to measure the amount of Ang, Angptl, or Tie specifically bound by    the coated anti-Mg, Angptl, or Tie amino acid sequence (in this case    Nanobody-X). The background signal for the assay is defined as the    signal obtained in wells with the coated amino acid sequence (in    this case Nanobody-X), second solution phase amino acid sequence (in    this case Nanobody-Y), Ang, Angptl, or Tie buffer only (i.e. no Ang,    Angptl, or Tie) and Ang, Angptl, or Tie detection reagents. The    positive control signal for the assay is defined as the signal    obtained in wells with the coated amino acid sequence (in this case    Nanobody-X), second solution phase amino acid sequence buffer only    (i.e. no second solution phase amino acid sequence), Ang, Angptl, or    Tie and Ang, Angptl, or Tie detection reagents. The ELISA assay may    be run in such a manner so as to have the positive control signal be    at least 6 times the background signal. To avoid any artefacts (e.g.    significantly different affinities between Nanobody-X and Nanobody-Y    for Ang, Angptl, or Tie) resulting from the choice of which amino    acid sequence to use as the coating amino acid sequence and which to    use as the second (competitor) amino acid sequence, the    cross-blocking assay may to be run in two formats: 1) format 1 is    where Nanobody-X is the amino acid sequence that is coated onto the    ELISA plate and Nanobody-Y is the competitor amino acid sequence    that is in solution and 2) format 2 is where Nanobody-Y is the amino    acid sequence that is coated onto the ELISA plate and Nanobody-X is    the competitor amino acid sequence that is in solution. Nanobody-X    and Nanobody-Y are defined as cross-blocking if, either in format 1    or in format 2, the solution phase anti-Ang, Angptl, or Tie amino    acid sequence is able to cause a reduction of between 60% and 100%,    specifically between 70% and 100%, and more specifically between 80%    and 100%, of the Ang, Angptl, or Tie detection signal {i.e. the    amount of Ang, Angptl, or Tie bound by the coated amino acid    sequence) as compared to the Ang, Angptl, or Tie detection signal    obtained in the absence of the solution phase anti-Ang, Angptl, or    Tie amino acid sequence (i.e. the positive control wells). An    example of such an ELISA-based cross blocking assay can be found in    Example [xxx] (“ELISA-based cross-blocking assay”).-   p) As further described herein, the total number of amino acid    residues in a Nanobody can be in the region of 110-120, is    preferably 112-115, and is most preferably 113. It should however be    noted that parts, fragments, analogs or derivatives (as further    described herein) of a Nanobody are not particularly limited as to    their length and/or size, as long as such parts, fragments, analogs    or derivatives meet the further requirements outlined herein and are    also preferably suitable for the purposes described herein;-   q) The amino acid residues of a Nanobody are numbered according to    the general numbering for V_(H) domains given by Kabat et al.    (“Sequence of proteins of immunological interest”, US Public Health    Services, NIH Bethesda, Md., Publication No. 91), as applied to    V_(HH) domains from Camelids in the article of Riechmann and    Muyldermans, J. Immunol. Methods 2000 Jun. 23; 240 (1-2): 185-195    (see for example FIG. 2 of this publication); or referred to herein.    According to this numbering, FR1 of a Nanobody comprises the amino    acid residues at positions 1-30, CDR1 of a Nanobody comprises the    amino acid residues at positions 31-35, FR2 of a Nanobody comprises    the amino acids at positions 36-49, CDR2 of a Nanobody comprises the    amino acid residues at positions 50-65. FR3 of a Nanobody comprises    the amino acid residues at positions 66-94, CDR3 of a Nanobody    comprises the amino acid residues at positions 95-102, and FR4 of a    Nanobody comprises the amino acid residues at positions 103-113. [In    this respect, it should be noted that—as is well known in the art    for V_(H) domains and for V_(HH) domains—the total number of amino    acid residues in each of the CDR's may vary and may not correspond    to the total number of amino acid residues indicated by the Kabat    numbering (that is, one or more positions according to the Kabat    numbering may not be occupied in the actual sequence, or the actual    sequence may contain more amino acid residues than the number    allowed for by the Kabat numbering). This means that, generally, the    numbering according to Kabat may or may not correspond to the actual    numbering of the amino acid residues in the actual sequence.    Generally, however, it can be said that, according to the numbering    of Kabat and irrespective of the number of amino acid residues in    the CDR's, position 1 according to the Kabat numbering corresponds    to the start of FR1 and vice versa, position 36 according to the    Kabat numbering corresponds to the start of FR2 and vice versa,    position 66 according to the Kabat numbering corresponds to the    start of FR3 and vice versa, and position 103 according to the Kabat    numbering corresponds to the start of FR4 and vice versa.].    -   Alternative methods for numbering the amino acid residues of        V_(H) domains, which methods can also be applied in an analogous        manner to V_(HH) domains from Camelids and to Nanobodies, are        the method described by Chothia et al. (Nature 342, 877-883        (1989)), the so-called “AbM definition” and the so-called        “contact definition”. However, in the present description,        claims and figures, the numbering according to Kabat as applied        to V_(HH) domains by Riechmann and Muyldermans will be followed,        unless indicated otherwise;-   r) In respect of a target or antigen, the term “interaction site” on    the target or antigen means a site, epitope, antigenic determinant,    part, domain or stretch of amino acid residues on the target or    antigen that is a site for binding to a ligand, receptor or other    binding partner, a catalytic site, a cleavage site, a site for    allosteric interaction, a site involved in multimerisation (such as    homomerization or heterodimerization) of the target or antigen; or    any other site, epitope, antigenic determinant, part, domain or    stretch of amino acid residues on the target or antigen that is    involved in a biological action or mechanism of the target or    antigen. More generally, an “interaction site” can be any site,    epitope, antigenic determinant, part, domain or stretch of amino    acid residues on the target or antigen to which an amino acid    sequence or polypeptide of the invention can bind such that the    target or antigen (and/or any pathway, interaction, signalling,    biological mechanism or biological effect in which the target or    antigen is involved) is modulated (as defined herein); and-   s) The Figures, Sequence Listing and the Experimental Part/Examples    are only given to further illustrate the invention and should not be    interpreted or construed as limiting the scope of the invention    and/or of the appended claims in any way, unless explicitly    indicated otherwise herein.

For a general description of heavy chain antibodies and the variabledomains thereof, reference is inter alia made to the prior art citedherein, to the review article by Muyldermans in Reviews in MolecularBiotechnology 74 (2001), 277-302; as well as to the following patentapplications, which are mentioned as general background art: WO94/04678, WO 95/04079 and WO 96/34103 of the Vrije Universiteit Brussel;WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO01/40310, WO 01/44301, EP 1134231 and WO 02/48193 of Unilever; WO97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 ofthe Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 ofAlgonomics N.V. and Ablynx N.V.; WO 01/90190 by the National ResearchCouncil of Canada; WO 03/025020 EP 1 433 793) by the Institute ofAntibodies; as well as WO 04/041867, WO 04/041862, WO 04/041865, WO04/041863, WO 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO06/122786, WO 06/122787 and WO 06/122825, by Ablynx N.V. and the furtherpublished patent applications by Ablynx N.V. Reference is also made tothe further prior art mentioned in these applications, and in particularto the list of references mentioned on pages 41-43 of the Internationalapplication WO 06/040153, which list and references are incorporatedherein by reference.

In accordance with the terminology used in the art (see the abovereferences), the variable domains present in naturally occurring heavychain antibodies will also be referred to as “V_(HH) domains”, in orderto distinguish them from the heavy chain variable domains that arepresent in conventional 4-chain antibodies (which will be referred toherein below as “V_(H) domains”) and from the light chain variabledomains that are present in conventional 4-chain antibodies (which willbe referred to herein below as “V_(L) domains”).

As mentioned in the prior art referred to above, V_(HH) domains have anumber of unique structural characteristics and functional propertieswhich make isolated V_(HH) domains (as well as Nanobodies based thereon,which share these structural characteristics and functional propertieswith the naturally occurring V_(HH) domains) and proteins containing thesame highly advantageous for use as functional antigen-binding domainsor proteins. In particular, and without being limited thereto, V_(HH)domains (which have been “designed” by nature to functionally bind to anantigen without the presence of, and without any interaction with, alight chain variable domain) and Nanobodies can function as a single,relatively small, functional antigen-binding structural unit, domain orprotein. This distinguishes the V_(HH) domains from the V_(H) and V_(L)domains of conventional 4-chain antibodies, which by themselves aregenerally not suited for practical application as single antigen-bindingproteins or domains, but need to be combined in some form or another toprovide a functional antigen-binding unit (as in for exampleconventional antibody fragments such as Fab fragments; in ScFv'sfragments, which consist of a V_(H) domain covalently linked to a V_(L)domain).

Because of these unique properties, the use of V_(HH) domains andNanobodies as single antigen-binding proteins or as antigen-bindingdomains (i.e. as part of a larger protein or polypeptide) offers anumber of significant advantages over the use of conventional V_(H) andV_(L) domains, scFv's or conventional antibody fragments (such as Fab-or F(ab′)₂-fragments):

-   -   only a single domain is required to bind an antigen with high        affinity and with high selectivity, so that there is no need to        have two separate domains present, nor to assure that these two        domains are present in the right special conformation and        configuration (i.e. through the use of especially designed        linkers, as with scFv's);    -   V_(HH) domains and Nanobodies can be expressed from a single        gene and require no post-translational folding or modifications;    -   V_(HH) domains and Nanobodies can easily be engineered into        multivalent and multispecific formats (as further discussed        herein);    -   V_(HH) domains and Nanobodies are highly soluble and do not have        a tendency to aggregate (as with the mouse-derived “dAb's”        described by Ward et al., Nature, Vol. 341, 1989, p. 544);    -   V_(HH) domains and Nanobodies are highly stable to heat, pH,        proteases and other denaturing agents or conditions (see for        example Ewert et al, supra);    -   V_(HH) domains and Nanobodies are easy and relatively cheap to        prepare, even on a scale required for production. For example,        V_(HH) domains, Nanobodies and proteins/polypeptides containing        the same can be produced using microbial fermentation (e.g. as        further described below) and do not require the use of mammalian        expression systems, as with for example conventional antibody        fragments;    -   V_(HH) domains and Nanobodies are relatively small        (approximately 1.5 kDa, or 10 times smaller than a conventional        IgG) compared to conventional 4-chain antibodies and        antigen-binding fragments thereof, and therefore show high(er)        penetration into tissues (including but not limited to solid        tumours and other dense tissues) than such conventional 4-chain        antibodies and antigen-binding fragments thereof;    -   V_(HH) domains and Nanobodies can show so-called cavity-binding        properties (inter alia due to their extended CDR3 loop, compared        to conventional V_(H) domains) and can therefore also access        targets and epitopes not accessible to conventional 4-chain        antibodies and antigen-binding fragments thereof. For example,        it has been shown that V_(HH) domains and Nanobodies can inhibit        enzymes (see for example WO 97/49805; Transue et al., Proteins        1998 Sep. 1; 32(4): 515-22; Lauwereys et al., EMBO J. 1998 Jul.        1; 17(13): 3512-20).

In a specific and preferred aspect, the invention provides Nanobodiesagainst Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3,Angptl4, Angptl5, or Angptl6, more preferably Tie2, Ang2, Ang1, Ang4, orAngptl4, more preferably Tie2 or Ang2; and in particular Nanobodiesagainst Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3,Angptl4, Angptl5, or Angptl6, more preferably Tie2, Ang2, Ang1, Ang4, orAngptl4, more preferably Tie2 or Ang2 from a warm-blooded animal, andmore in particular Nanobodies against Tie1, Tie2, Ang1, Ang2, Ang3,Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, morepreferably Tie2, Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2 orAng2 from a mammal, and especially Nanobodies against human Tie1, Tie2,Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, morepreferably Tie2 or Ang2; as well as proteins and/or polypeptidescomprising at least one such Nanobody.

In particular, the invention provides Nanobodies against Tie1, Tie2,Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, morepreferably Tie2 or Ang2, and proteins and/or polypeptides comprising thesame, that have improved therapeutic and/or pharmacological propertiesand/or other advantageous properties (such as, for example, improvedease of preparation and/or reduced costs of goods), compared toconventional antibodies against Tie1, Tie2, Ang1, Ang2, Ang3, Ang4,Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, more preferablyTie2, Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2 or Ang2 orfragments thereof, compared to constructs that could be based on suchconventional antibodies or antibody fragments (such as Fab′ fragments,F(ab′)₂ fragments, ScFv constructs, “diabodies” and other multispecificconstructs (see for example the review by Holliger and Hudson, NatBiotechnol. 2005 September; 23(9):1126-36)), and also compared to theso-called “dAb's” or similar (single) domain antibodies that may bederived from variable domains of conventional antibodies. These improvedand advantageous properties will become clear from the furtherdescription herein, and for example include, without limitation, one ormore of:

-   -   increased affinity and/or avidity for Tie1, Tie2, Ang1, Ang2,        Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or        Angptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4,        more preferably Tie2 or Ang2, either in a monovalent format, in        a multivalent format (for example in a bivalent format) and/or        in a multispecific format (for example one of the multispecific        formats described herein below);    -   better suitability for formatting in a multivalent format (for        example in a bivalent format);    -   better suitability for formatting in a multispecific format (for        example one of the multispecific formats described herein        below); improved suitability or susceptibility for “humanizing”        substitutions (as defined herein);    -   less immunogenicity, either in a monovalent format, in a        multivalent format (for example in a bivalent format) and/or in        a multispecific format (for example one of the multispecific        formats described herein below);    -   increased stability, either in a monovalent format, in a        multivalent format (for example in a bivalent format) and/or in        a multispecific format (for example one of the multispecific        formats described herein below);    -   increased specificity towards any of Tie1, Tie2, Ang1, Ang2,        Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or        Angptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4,        more preferably Tie2 or Ang2, either in a monovalent format, in        a multivalent format (for example in a bivalent format) and/or        in a multispecific format (for example one of the multispecific        formats described herein below);    -   decreased or where desired increased cross-reactivity with any        of Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,        Angptl3, Angptl4, Angptl5, or Angptl6, more preferably Tie2,        Ang2, Ang1 Ang4, or Angptl4, more preferably Tie2 or Ang2 from        different species;        and/or    -   one or more other improved properties desirable for        pharmaceutical use (including prophylactic use and/or        therapeutic use) and/or for diagnostic use (including but not        limited to use for imaging purposes), either in a monovalent        format, in a multivalent format (for example in a bivalent        format) and/or in a multispecific format (for example one of the        multispecific formats described herein below).

As generally described herein for the amino acid sequences of theinvention, the Nanobodies of the invention are preferably in essentiallyisolated form (as defined herein), or form part of a protein orpolypeptide of the invention (as defined herein), which may comprise oressentially consist of one or more Nanobodies of the invention and whichmay optionally further comprise one or more further amino acid sequences(all optionally linked via one or more suitable linkers). For example,and without limitation, the one or more amino acid sequences of theinvention may be used as a binding unit in such a protein orpolypeptide, which may optionally contain one or more further amino acidsequences that can serve as a binding unit (i.e. against one or moreother targets than Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,Angptl3, Angptl4, Angptl5, or Angptl6, more preferably Tie2, Ang2, Ang1,Ang4, or Angptl4, more preferably Tie2 or Ang2), so as to provide amonovalent, multivalent or multispecific polypeptide of the invention,respectively, all as described herein. In particular, such a protein orpolypeptide may comprise or essentially consist of one or moreNanobodies of the invention and optionally one or more (other)Nanobodies (i.e. directed against other targets than Tie1, Tie2, Ang1,Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, morepreferably Tie2 or Ang2), all optionally linked via one or more suitablelinkers, so as to provide a monovalent, multivalent or multispecificNanobody construct, respectively, as further described herein. Suchproteins or polypeptides may also be in essentially isolated form (asdefined herein).

In a Nanobody of the invention, the binding site for binding againstTie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4,Angptl5, or Angptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4,more preferably Tie2 or Ang2 is preferably formed by the CDR sequences.Optionally, a Nanobody of the invention may also, and in addition to theat least one binding site for binding against Tie1, Tie2, Ang1, Ang2,Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6,more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2or Ang2, contain one or more further binding sites for binding againstother antigens, proteins or targets. For methods and positions forintroducing such second binding sites, reference is for example made toKeck and Huston, Biophysical. Journal, 71, October 1996, 2002-2011; EP 0640 130; WO 06/07260 and the US provisional application by Ablynx N.V,entitled “Immunoglobulin domains with multiple binding sites” filed onNov. 27, 2006.

As generally described herein for the amino acid sequences of theinvention, when a Nanobody of the invention (or a polypeptide of theinvention comprising the same) is intended for administration to asubject (for example for therapeutic and/or diagnostic purposes asdescribed herein), it is preferably directed against human Tie1, Tie2,Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, morepreferably Tie2 or Ang2; whereas for veterinary purposes, it ispreferably directed against Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1,Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, more preferably Tie2,Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2 or Ang2 from thespecies to be treated. Also, as with the amino acid sequences of theinvention, a Nanobody of the invention may or may not be cross-reactive(i.e. directed against Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1,Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, more preferably Tie2,Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2 or Ang2 from two ormore species of mammal, such as against human Tie1, Tie2, Ang1, Ang2,Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6,more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2or Ang2 and Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,Angptl3, Angptl4, Angptl5, or Angptl6, more preferably Tie2, Ang2, Ang1,Ang4, or Angptl4, more preferably Tie2 or Ang2 from at least one of thespecies of mammal mentioned herein).

Also, again as generally described herein for the amino acid sequencesof the invention, the Nanobodies of the invention may generally bedirected against any antigenic determinant, epitope, part, domain,subunit or confirmation (where applicable) of Tie1, Tie2, Ang1, Ang2,Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6,more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2or Ang2. However, it is generally assumed and preferred that theNanobodies of the invention (and polypeptides comprising the same) aredirected against the binding site of Ang1 on Tie2 or the binding site ofTie2 on Ang2.

As already described herein, the amino acid sequence and structure of aNanobody can be considered—without however being limited thereto—to becomprised of four framework regions or “FR's” (or sometimes alsoreferred to as “FW's”), which are referred to in the art and herein as“Framework region 1” or “FR1.”; as “Framework region 2” or “FR2”; as“Framework region 3” or “FR3”; and as “Framework region 4” or “FR4”,respectively; which framework regions are interrupted by threecomplementary determining regions or “CDR's”, which are referred to inthe art as “Complementarity Determining Region 1” or “CDR1”; as“Complementarity Determining Region 2” or “CDR2”; and as“Complementarity Determining Region 3” or “CDR3”, respectively. Somepreferred framework sequences and CDR's (and combinations thereof) thatare present in the Nanobodies of the invention are as described herein.Other suitable CDR sequences can be obtained by the methods describedherein.

According to a non-limiting but preferred aspect of the invention, (theCDR sequences present in) the Nanobodies of the invention are such that:

-   -   the Nanobodies can bind to Tie1, Tie2, Ang1, Ang2, Ang3, Ang4,        Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, more        preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, more preferably        Tie2 or Ang2 with a dissociation constant (K_(D)) of 10⁻⁵ to        10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²        moles/liter or less and more preferably 10⁻⁸ to 10⁻¹²        moles/liter (i.e. with an association constant (K_(A)) of 10⁵ to        10¹² liter/moles or more, and preferably 10⁷ to 10¹² liter/moles        or more and more preferably 10⁸ to 10¹² liter/moles);        and/or such that:    -   the Nanobodies can bind to Tie1, Tie2, Ang1, Ang2, Ang3, Ang4,        Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, more        preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, more preferably        Tie2 or Ang2 with a k_(on)-rate of between 10² M⁻¹s⁻¹ to about        10⁷ M⁻¹s⁻¹, preferably between 10³ M⁻¹s⁻¹ and 10⁷ s⁻¹, more        preferably between 10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between        10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹;        and/or such that they:    -   the Nanobodies can bind to Tie1, Tie2, Ang1, Ang2, Ang3, Ang4,        Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, more        preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, more preferably        Tie2 or Ang2 with a k_(off) rate between 1s⁻¹ (t_(1/2)=0.69 s)        and 10⁻⁶ s⁻¹ (providing a near irreversible complex with a        t_(1/2) of multiple days), preferably between 10⁻² s⁻¹ and 10⁻⁶        s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as        between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, (the CDR sequences present in) the Nanobodies of theinvention are such that: a monovalent Nanobody of the invention (or apolypeptide that contains only one Nanobody of the invention) ispreferably such that it will bind to Tie1, Tie2, Ang1, Ang2, Ang3, Ang4,Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, more preferablyTie2, Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2 or Ang2 with anaffinity less than 500 nM, preferably less than 200 nM, more preferablyless than 10 nM, such as less than 500 pM.

The affinity of the Nanobody of the invention against Tie1, Tie2, Ang1,Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, morepreferably Tie2 or Ang2 can be determined in a manner known per se, forexample using the general techniques for measuring K_(D). K_(A), k_(off)or k_(on) mentioned herein, as well as some of the specific assaysdescribed herein.

Some preferred IC50 values for binding of the Nanobodies of theinvention (and of polypeptides comprising the same) to Tie1, Tie2, Ang1,Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, morepreferably Tie2 or Ang2 will become clear from the further descriptionand examples herein.

In a preferred but non-limiting aspect, the invention relates to aNanobody (as defined herein) against Tie1, Tie2, Ang1, Ang2, Ang3, Ang4,Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, more preferablyTie2, Ang2, Ang1 Ang4, or Angptl4, more preferably Tie2 or Ang2, whichconsists of 4 framework regions (FR1 to FR4 respectively) and 3complementarity determining regions (CDR1 to CDR3 respectively), inwhich:

CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 173 to 219;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 173 to    219;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 173 to    219;    and/or

CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 267 to 313;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 267 to    313;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 267 to    313;    and/or

CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 361 to 454;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 361 to    454;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 361 to    454;    or any suitable fragment of such an amino acid sequence.

In particular, according to this preferred but non-limiting aspect, theinvention relates to a Nanobody (as defined herein) against Tie1, Tie2,Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, morepreferably Tie2 or Ang2, which consists of 4 framework regions (FR1 toFR4 respectively) and 3 complementarity determining regions (CDR1 toCDR3 respectively), in which:

CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 173 to 219;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 173 to    219;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 173 to    219;    and

CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 267 to 313;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 267 to    313;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 267 to    313;    and

CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 361 to 454;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 361 to    454;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 361 to    454;    or any suitable fragment of such an amino acid sequences.

As generally mentioned herein for the amino acid sequences of theinvention, when a Nanobody of the invention contains one or more CDR1sequences according to b) and/or c):

-   i) any amino acid substitution in such a CDR according to b)    and/or c) is preferably, and compared to the corresponding CDR    according to a), a conservative amino acid substitution (as defined    herein);    and/or-   ii) the CDR according to b) and/or c) preferably only contains amino    acid substitutions, and no amino acid deletions or insertions,    compared to the corresponding CDR according to a);    and/or-   iii) the CDR according to b) and/or c) may be a CDR that is derived    from a CDR according to a) by means of affinity maturation using one    or more techniques of affinity maturation known per se.

Similarly, when a Nanobody of the invention contains one or more CDR2sequences according to e) and/or f):

-   i) any amino acid substitution in such a CDR according to e)    and/or f) is preferably, and compared to the corresponding CDR    according to d), a conservative amino acid substitution (as defined    herein);    and/or-   ii) the CDR according to e) and/or f) preferably only contains amino    acid substitutions, and no amino acid deletions or insertions,    compared to the corresponding CDR according to d);    and/or-   iii) the CDR according to e) and/or f) may be a CDR that is derived    from a CDR according to d) by means of affinity maturation using one    or more techniques of affinity maturation known per se.

Also, similarly, when a Nanobody of the invention contains one or moreCDR3 sequences according to h) and/or i):

-   i) any amino acid substitution in such a CDR according to h)    and/or i) is preferably, and compared to the corresponding CDR    according to g), a conservative amino acid substitution (as defined    herein);    and/or-   ii) the CDR according to h) and/or 1) preferably only contains amino    acid substitutions, and no amino acid deletions or insertions,    compared to the corresponding CDR according to g);    and/or-   iii) the CDR according to h) and/or i) may be a CDR that is derived    from a CDR according to g) by means of affinity maturation using one    or more techniques of affinity maturation known per se.

It should be understood that the last three paragraphs generally applyto any Nanobody of the invention that comprises one or more CDR1sequences, CDR2 sequences and/or CDR3 sequences according to b), c), e),h) or i), respectively.

Of the Nanobodies of the invention, Nanobodies comprising one or more ofthe CDR's explicitly listed above are particularly preferred; Nanobodiescomprising two or more of the CDR's explicitly listed above are moreparticularly preferred; and Nanobodies comprising three of the CDR'sexplicitly listed above are most particularly preferred.

Some particularly preferred, but non-limiting combinations of CDRsequences, as well as preferred combinations of CDR sequences andframework sequences, are mentioned in Table A-1 below, which lists theCDR sequences and framework sequences that are present in a number ofpreferred (but non-limiting) Nanobodies of the invention. As will beclear to the skilled person, a combination of CDR1, CDR2 and CDR3sequences that occur in the same clone (i.e. CDR1, CDR2 and CDR3sequences that are mentioned on the same line in Table A-1) will usuallybe preferred (although the invention in its broadest sense is notlimited thereto, and also comprises other suitable combinations of theCDR sequences mentioned in Table A-1). Also, a combination of CDRsequences and framework sequences that occur in the same clone (i.e. CDRsequences and framework sequences that are mentioned on the same line inTable A-1) will usually be preferred (although the invention in itsbroadest sense is not limited thereto, and also comprises other suitablecombinations of the CDR sequences and framework sequences mentioned inTable A-1, as well as combinations of such CDR sequences and othersuitable framework sequences, e.g. as further described herein).

Also, in the Nanobodies of the invention that comprise the combinationsof CDR's mentioned in Table A-1, each CDR can be replaced by a CDRchosen from the group consisting of amino acid sequences that have atleast 80%, preferably at least 90%, more preferably at least 95%, evenmore preferably at least 99% sequence identity (as defined herein) withthe mentioned CDR's; in which:

-   i) any amino acid substitution in such a CDR is preferably, and    compared to the corresponding CDR sequence mentioned in Table A-1, a    conservative amino acid substitution (as defined herein);    and/or-   ii) any such CDR sequence preferably only contains amino acid    substitutions, and no amino acid deletions or insertions, compared    to the corresponding CDR sequence mentioned in Table A-1;    and/or-   iii) any such CDR sequence is a CDR that is derived by means of a    technique for affinity maturation known per se, and in particular    starting from the corresponding CDR sequence mentioned in Table A-1.

However, as will be clear to the skilled person, the (combinations of)CDR sequences, as well as (the combinations of) CDR sequences andframework sequences mentioned in Table A-1 will generally be preferred.

TABLE A-1Preferred combinations of CDR sequences, preferred combinations of framework sequences,and preferred combinations of framework and CDR sequences. Clone ID FR1ID CDR1 ID FR2 ID CDR2 ID FR3 ID CDR3 ID FR4 ID 162- 455 EVQLVESGGGL 126INAMG 173 WYQQAPGKQ 226 FITSVG 267 RFIISRDNAKNTVYLQ 314 DLHYSGPN 361WGQGTQVTV 408 E1 VQAGGSLRLSC RELVA TTNYAD MNSLKPEDTAVYYCA Y SS AASGSIFSSVKG A 162- 456 EVQLVESGGGL 127 DYAIG 174 WFRQAPGKE 221 CISSVD 268RFTISRDNAKDTVYL 315 QGYSGGYY 362 WGQGTQVTV 409 E9 VQPGGSLRLSC REAVSGSTHYA QMNSLKPEDTAAYYC YTCEDSAD SS AASGFTLD DSVKG AV FGF 162- 457EVQLVESGGGL 128 DYAIG 175 WFRQAPGKE 222 CISSSD 269 RFTISSDNAKNTVYLQ 316GSVAGCIP 363 WGQGTQVTV 410 F11 VQAGGSLRLSC  REGVA GSTYYA SSMNSLKPEDTAVYSCS YY AASGFTLD DSVKG A 162- 458 EVQLVESGGGL 129 DDTMG 176WFRQAPRKE 223 AILWDS 270 RFTISRDNAKNTVYL 317 TPTAYGTD 364 WGQGTQVTV 411F3 VQAGDSLRLSCT REFVA IKTYYA QMDSLKPEDTAVYYC WYRNNYHY SS TSGRTFS DSVKGAA 162- 459 EVQLVESGGGL 130 DYAVG 177 WFRQAPGKE 224 CIGSSY 271RFTISRDNAKNTVYL 318 QGYSGGYY 365 WGQGTQVTV 412 H10 VQPGGSLRLSC REGVSGSTYYA QMNSLKPEDTAVYYC YTCEDSAD SS AASGFTLD     DSVKG AV FGF 163- 460EVQLVESGGGL 131 DYSMS 178 WVRQAPGKG 225 AISGGGE 272 RFTISRDNAKNTLYLQ 319HLNFYSVS 366 TSQGTQVTVS 413 E7 VQPGGSLRLSC LEWVS VTTYADS MSSLKPEDTALYYCAVRSSP S AASGFTFS VKG E 163- 461 EVQLVESGGGL 132 SNGMR 179 WVRQAPGKG 226SINSDGT 273 RFTISRDNAKNTLCLQ 320 TEDPYP 367 RGQGTQVTV 414 E9 VQPGDSLRLSCPEWVS STYYADS MNSLKPEDTAVYYC SS AASGFTFG VKG T 163- 462 VQLVESGGGL 133SNGMR 180 WVRQAPGKG 227 SINSDGT 274 RFTISRDNAKNTLYLQ 321 TMNPNP 368RGQGTQVTV 415 GB VQPGGSLRLSC PEWVS SAFYAES MNSLKPEDTAVYYCT SS AASGFTFGVKG T 163- 463 EVQLVESGGGL 134 SNGMR 181 WVRQAPGKG 228 SINSDGT 275RFTISRDNAKNTLYLQ 322 TENPNP 369 RGPGTQVTVS 416 H8 VQPGGSLRLSC PEWVSSTYYAES MHSLKPEDTAVYYCT S AASGFTFG VKG T 166- 464 EVQLVESGGGL 135 STTIG182 WFRQAPGKE 229 CISTGDG 276 RFTISSDNAKNTVYLQ 323 DQAPMWS 370 WGQGTQVTV417 C1 VQAGGSLRLSC REGVS STYYAES MNSLKPEDTAVYYCA SWSAPYEY SS AASGFTFGVKG L DY 166- 465 EVQLVESGGGL 136 TTTLG 183 WFRQAPGKE 230 CISTGDG 277RFTISSDNAKNTVYLQ 324 DQAPMWS 371 WGQGTQVTV 418 C10 VQAGGSLRLSC REGVSSTNYAES MNSLKPEDTAVYYCA SWSAPYEY SS AASGFTFG VKG L DY 166- 466EVQLVESGGGL 137 DTTIG 184 WFRQAPGKE 231 CISTGDG 278 RFTISSDNAKNTVYLQ 325DQAPLWST 372 WGQGTQVTV 419 D7 VQAGGSLRLSC REGIS STYYAES MNSLNPEDTAVYYCAWSAPYEYD SS AASGFTFS VKG L Y 166- 467 EVQLVESGGGL 138 TTTIG 185WFRQAPGKE 232 CISTGGG 279 RFTISSDNAKNTVYLQ 326 DQAPMWS 373 WGQGTQVTV 420F8 VQAGGSLRLSC REVVS TYYTESV MNSLKPEDTAVYYCA NWSAPYEY SS AASGFTFG KG LDY 166- 468 EVQLVESGGGL 139 DTTIG 186 WFRQAPGKE 233 CISTGDG 280RFTISSDNAKNTVYLQ 327 DQAPLWST 374 WGQGTQVTV 421 G4 VQAGGALRLSC REGISSTYYAES MNSLNPEDTAVYYCA WSAPYEYD SS AASGFTFS VKG L Y 166- 469EVQLVESGGDL 140 DFTIG 187 WFRQAPGKE 234 CINTGDG 281 RFTISSDNAKNTVYLQ 328DQAPMWS 375 WGQGTQVTV 422 H4 VQAGGSLRLSC REGVS STNYAES MNSLKPEDTAVYYCASWSAPYEY SS AASGFTFG VKG L DY 166- 470 KVQLVESGGGL 141 STTIG 188WFRQAPGKE 235 CISTGDG 282 RFTISSDNAKNTVYLQ 329 DQAPMWS 376 WGQGTQVTV 423E12 VQAGGSLRLSC REGVS STYYAES MNSLKPEDTAVYYCA SWSAPYEY SS AASGFTFG VKG LDY 166- 471 EVQLVESGGGL 142 NTAMG 189 WYRQAPGKW 236 TIYSGGS 283RFIISRDNTRNTVHLQ 330 VGAGSY 377 WGQGAQVTV 424 D4 VQAGGSLRLSC RELVATKYIDSV MNSLKPEDTAVYYCN SS VASGRIFT KG T 173- 472 EVQLVESGGGL 143 GNWMY190 WLRQAPGKG 237 TITPRGL 284 RFTISRDIAENTLYLQ 331 DKTGER 378 RGQGTQVTV425 H9 VQPGGSLRLSC LEWIS TAYADSV MNSLKSGDTAVYYCA SS AASGFTLS KG R 184-476 EVQLVESGGGL 144 NYAMT 191 WVRQAPGKG 238 DISWDGD 285 RFTISRDNAKKTLYLQ332 YGYDSGRY 379 WGQGTQVTV 426 B6 VQPGGSLRLSC LEWVS ITTYAASMNSLKPEDSAVYYCN YSY SS AASGFTFS VKG T 185- 474 EVQLVESGGGL 145 YYAIG 192WFRQAPGKE 239 YISSSDG 286 RFTISRDNAKNTVYL 333 DLSGRGDV 380 WGQGTQVTV 427H5 VQPGGSLRLSC REGVS RTYYADS QMNSLKPEDTAVYYC SEYEYDY SS AASGFTLD VKG AT168- 475 EVQLVESGGGL 146 GNWMY 193 WLRQAPGKG 240 TITPRGL 287RFTISRDIAENTLYLQ 334 DKTGER 381 RGQGTQVTV 428 A3 VQPGGSLRLSC LEWISTAYADSV MNSLKSGDTAVYYCA SS AASGFTLS KG R 168- 476 EVQLVESGGGL 147 SNWMY194 WLRQAPGKG 241 TITPRDL 288 RFTISRDNAENTLYLQ 335 DKAGER 382 RGQGTQVTV429 E5 VQPGGSLRLSC LEWIS TAYADSV MNSLKSEDTAVYYCA SS AASGFTLS KG K 168-477 EVQLVESGGGL 148 YYAIG 195 WYRQAPGKE 242 CISSSNY 289 RFTISRDNAKNTVYL336 NTRRKYGR 383 WGQGTQVTV 430 G3 VQPGGSLRLSC REWVS GITTYADQMNSLKPEDTAIYYC LCDLNADY SS AASGSTLD SVKG AT 169- 478 EVQLVESGGGL 149PSWMY 196 WLRQAPGKG 243 TITPRGL 290 RFTISKDNAKNTLYLQ 337 DKNGPP 384MGQGTQVTV 431 A10 VQPGGSLRLSC LEWVS TEYANSV MNSLKSEDTAVYYCT SS ATSGFTFSKG R 169- 479 EVQLVESGGGL 150 IIHMG 197 WYRQAPGNE 244 VIIDSRT 291RFTISRDNAKNTVYL 338 LALGTDQS 385 WGQGTQVTV 432 A12 VQPGGSLRLSC RDLVATKYSESV QMNSLKPEDTAVYYC STFDS SS VASGSIRS KG NA 169- 480 EVQLVESGGGL 151INAMG 198 WYRQAPGNQ 245 AITSGDS 292 RFTISRDNAKNTVYL 339 ELLGKWY 386WGQGTQVTV 433 B12 VQAGGSLRLSC RDLVA TKYADFV QMNSLKPEDTAVYYC SS AASGSIFSKG AA 169- 481 EVQLVESGGGL 152 IIHMG 199 WYRQTPGNE 246 VIISRTT 293RFTISRDNAKNTVYL 340 LALGTDQS 387 WGQGTQVTV 434 C12 VQPGGSLRLSC RDMVAKYAESVK QMNSLKPEDTAVYYC STFDS SS AASGSIRS G NA 169- 482 WVQLVESGGGL 153TSWMY 200 WLRQAPGKG 247 TITPRGL 294 RFTISRDSAKNTLYLQ 341 DKNGPP 388MGQGTQVTV 435 C8 VQPGGSLRLSC LEWVS TDYTDSV MNSLKSEDTADYYCT SS ATSGFTFSKG R 169- 483 EVQLVESGGGL 154 INTMG 201 WYRQAPGNQ 248 AITNGGS 295RFTISRDNAKNTVYL 342 ESLGRWG 389 WGQGTQVTV 436 E12 VQAGGSLRLSC RDLVATKYVDSV QMNSLKPEDTAVYYC SS AASGSIFS KG AA 169- 484 EVQLVESGGGL 155 TSWMY202 WLRQAPGKG 249 TITPRGL 296 RFTVSRDNAKNTLYL 343 DKNGPP 390 MGQGTQVTV437 F11 VQPGGSLRLSC LEWVS TDYTNSV QMNSLKSEDTAVYYC SS ATSGFTFS KG TK 170-485 EVQLVESGGGL 156 LYVTG 203 WYRQAPGKQ 250 SITSGGS 297 RFTISRDNAKNTVHL344 RSIGVDDM 391 WGQGTQVTV 438 B1 VQAGGSLRLSC RELVA LTYADSVQMHSLKPEDTAVYFC PYVY SS AASESIFS KG NG 170- 486 EVQLVESGGGL 157 LNAMT204 WVRQAPGKQ 251 TISSGGW 298 RFTISRDNAKNTLYLQ 345 GSEFNGYE 392RGQGTQVTV 439 C2 VQPGGSLRLSC LEWVS TTSYADS MNSLKPEDMAVYYC V SS AASGFTFSVKG AK 170- 487 EVQLVESGGGL 158 INVMG 205 WYRQAPGKQ 252 TITRALN 299RFTISRDNFTNTVYLQ 346 GGYYTNLR 393 WGQGTQVTV 440 E2 VQAGGSLRLSC RDLVATAYATSV MNSLEPEDTAVYYCN TGGNY SS AASGSISS KG A 170- 488 EVQLVESGGGL 159DTMG 206 WYRQAPGKQ 253 SITPTGN 300 RFAISRDNNKNTMHL 347 VYPRYYGD 394WGQGTRVTV 441 F2 VQAGGSLRLSC RELVA TNYVDSV QMNSLKPEDTAVYYC DDRPPVDS SSAASGIFII KG NA 170- 489 EVQLVESGGGL 160 INVMG 207 WYRQAPGKQ 254 VITRALN301 RFTISRDDFKDTVYLQ 348 GGYYTNLR 395 WGQGTQVTV 442 H1 AQAGGSLRLSC RDLVATNYATSV MNSLEPEDTAVYYCN TGGNY SS AASGSISS KG A 171- 490 EVQLVESGGGQ 161TYGMG 208 WFRQAPGDK 255 SISASGA 302 RFTISRDNIKNTVYLQ 349 APNGRFIT 396WGQGTQVTV 443 A2 VQAGDSLRLSC RDLVS STYYVDS MNSLKPEDAAVYYCA MSAHVDS SSKASRRTIS VKG A 171- 491 EVQLVESGGGQ 162 TYGMG 209 WFRQAPGDK 256 SISASGA303 RFTISRDNIKNTVYLQ 350 APNGRFIT 397 WGQGTQVTV 444 A3 VQAGDSLRLSC RDLVSSTYYVDS MNSLKPEDAAVYYCA MSTHVDY SS KASRRTIS VKG A 171- 492 EVQLVESGGGL163 TFNTYS 210 WFRQAPGKE 257 AISRGGN 304 RFAISRDNAKNTVAL 351 SKIGIASTIR398 WGQGTQVTV 445 C4 VQPGGSLRLSC MG REFVA VTPYADS QMNSLKPEDAAVYYC YYDYSS AASGRTFS VKG AA 171- 493 EVQLVESGGGL 164 TYTVG 211 WFRQAPGKE 258IITGSGT 305 RFTVSRDNAKNTVYL 352 RHWGMFS 399 WGQGTQVTV 446 D2 VQAGGSLRLSCREFVS YNDYADS QMNSLKSEDAAVYYC RSENDYNY SS AASVLTFS VKG AA 171- 494EVQLVESGGGL 165 WYAMG 212 WFRQQAPGK 259 SSISGGG 306 RFTVSRDRAKNTVYL 353DKRWGSPA 400 WGQGTQVTV 447 E2 VQAGASLRLSCV EREFV SNTVYAD QMNSLKPEDSGVYYTSRSTHDY SS DSGDTFS SVKG CAA 171- 495 EVQLVESGGGL 166 TFNTYS 213WFRQAPGKE 260 AISRSGN 307 RFAISRDNAKNTLTLQ 354 SKIGIASTIR 401 WGQGTQVTV448 E4 VQPGGSLRLSC MG REFVA VTPYADS MNSLKPEDTAVYYCA YYDY SS AASGRTFS VKGA 171- 496 EVQLVESGGGL 167 LYYMG 214 WRQAPGRE 261 GISGSGG 308RFTISRDNLKNTMYL 355 SRRIITNPR 402 WGQGTQVTV 449 F3 VQTGGSLRLSC REFVASTFYGDS QMNSLKPEDTAVYYC EYGY SS AASGRSFN VKG QS 171- 497 EVQLVESGGGL 168MYAMA 215 WIRLAPGKER 262 AIDWSGG 309 RFTISRDNAKNTVYLE 356 NRRIYSSG 403WGQGTQVTV 450 G2 VQAGGSLRLSC EVIA STFYGDS MNSLKPEDTAVYYCA SSLSDNSL SSTASGLTFS VKG A YNF 171- 498 EVQLVESGGGL 169 WYAMG 216 WFRQQAPGK 263SAISGGG 310 RFAISRDNADSTYLR 357 DKRWGSPA 404 WGQGTQVTV 451 G4VQAGGSLRLSC EREFV SNIVYYD MNNLKPEDTAVYYCN TSRSTHDY SS VASGDTFN SVKG A DF170- 499 EVQLVESGGGL 170 SAMG 217 WYRQPPG 264 RITRGGS 311RFTISKDIAKNTVYLQ 358 DTIGHSSSY 405 WGQGTQVTV 452 G3 VQAGGSLRLSC RELVATNYAESV MNSLKPDDMAVYYC ITY SS AASETIFA KG AA 171- 500 EVQLVESGGGL 171MYMAG 218 WFRQAPGKE 265 VITWSGG 312 RFTISKDIAKNTVYLQ 259 ARRYGNLY 406WGQGTQVTV 453 G2 VQAGGSLRLSC REFVT STYYADS MNSLKPDDMAVYYC NTNNYDY SSAASGRPFS VKG AA 171- 501 EVQLVESGGGQ 172 TYGMG 219 WFRQAPGDK 266 SISASGA313 RFTISKDNIKNTVYLQ 360 APNGRFIT 407 WGQGTVTV 454 H4 VQAGDSLRLSC RDLVSSTYYVDS MNSLKPDDAAVYYCA MSTHVDS SS KASRRTIS VKG A (“ID” refers to theSEQ ID NO in the attached sequence listing)

Thus, in the Nanobodies of the invention, at least one of the CDR1. CDR2and CDR3 sequences present is suitably chosen from the group consistingof the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1;or from the group of CDR1, CDR2 and CDR3 sequences, respectively, thathave at least 80%, preferably at least 90%, more preferably at least95%, even more preferably at least 99% “sequence identity” (as definedherein) with at least one of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table A-1; and/or from the group consisting ofthe CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only1 “amino acid difference(s)” (as defined herein) with at least one ofthe CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1.

In this context, by “suitably chosen” is meant that, as applicable, aCDR1 sequence is chosen from suitable CDR1 sequences (i.e. as definedherein), a CDR2 sequence is chosen from suitable CDR2 sequences (i.e. asdefined herein), and a CDR3 sequence is chosen from suitable CDR3sequence (i.e. as defined herein), respectively. More in particular, theCDR sequences are preferably chosen such that the Nanobodies of theinvention bind to Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,Angptl3, Angptl4, Angptl5, or Angptl6, more preferably Tie2, Ang2, Ang1,Ang4, or Angptl4, more preferably Tie2 or Ang2 Tie1, Tie2, Ang1, Ang2,Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6,more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2or Ang2 with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein.

In particular, in the Nanobodies of the invention, at least the CDR3sequence present is suitably chosen from the group consisting of theCDR3 sequences listed in Table A-1 or from the group of CDR3 sequencesthat have at least 80%, preferably at least 90%, more preferably atleast 95%, even more preferably at least 99% sequence identity with atleast one of the CDR3 sequences listed in Table A-1; and/or from thegroup consisting of the CDR3 sequences that have 3, 2 or only 1 aminoacid difference(s) with at least one of the CDR3 sequences listed inTable A-1.

Preferably, in the Nanobodies of the invention, at least two of theCDR1, CDR2 and CDR3 sequences present are suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed inTable A-1 or from the group consisting of CDR1, CDR2 and CDR3 sequences,respectively, that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table A-1; and/or from the group consisting ofthe CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only1 “amino acid difference(s)” with at least one of the CDR1, CDR2 andCDR3 sequences, respectively, listed in Table A-1.

In particular, in the Nanobodies of the invention, at least the CDR3sequence present is suitably chosen from the group consisting of theCDR3 sequences listed in Table A-1 or from the group of CDR3 sequencesthat have at least 80%, preferably at least 90%, more preferably atleast 95%, even more preferably at least 99% sequence identity with atleast one of the CDR3 sequences listed in Table A-1, respectively; andat least one of the CDR1 and CDR2 sequences present is suitably chosenfrom the group consisting of the CDR1 and CDR2 sequences, respectively,listed in Table A-1 or from the group of CDR1 and CDR2 sequences,respectively, that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the CDR1 and CDR2 sequences, respectively,listed in Table A-1; and/or from the group consisting of the CDR1 andCDR2 sequences, respectively, that have 3, 2 or only 1 amino aciddifference(s) with at least one of the CDR1 and CDR2 sequences,respectively, listed in Table A-1.

Most preferably, in the Nanobodies of the invention, all three CDR1,CDR2 and CDR3 sequences present are suitably chosen from the groupconsisting of the CDR1. CDR2 and CDR3 sequences, respectively, listed inTable A-1 or from the group of CDR1, CDR2 and CDR3 sequences,respectively, that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table A-1; and/or from the group consisting ofthe CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only1 amino acid difference(s) with at least one of the CDR1. CDR2 and CDR3sequences, respectively, listed in Table A-1.

Even more preferably, in the Nanobodies of the invention, at least oneof the CDR1, CDR2 and CDR3 sequences present is suitably chosen from thegroup consisting of the CDR1, CDR2 and CDR3 sequences, respectively,listed in Table A-1. Preferably, in this aspect, at least one orpreferably both of the other two CDR sequences present are suitablychosen from CDR sequences that have at least 80%, preferably at least90%, more preferably at least 95%, even more preferably at least 99%sequence identity with at least one of the corresponding CDR sequences,respectively, listed in Table A-1; and/or from the group consisting ofthe CDR sequences that have 3, 2 or only 1 amino acid difference(s) withat least one of the corresponding sequences, respectively, listed inTable A-1.

In particular, in the Nanobodies of the invention, at least the CDR3sequence present is suitably chosen from the group consisting of theCDR3 listed in Table A-1. Preferably, in this aspect, at least one andpreferably both of the CDR1 and CDR2 sequences present are suitablychosen from the groups of CDR1 and CDR2 sequences, respectively, thathave at least 80%, preferably at least 90%, more preferably at least95%, even more preferably at least 99% sequence identity with the CDR1and CDR2 sequences, respectively, listed in Table A-1; and/or from thegroup consisting of the CDR1 and CDR2 sequences, respectively, that have3, 2 or only 1 amino acid difference(s) with at least one of the CDR1and CDR2 sequences, respectively, listed in Table A-1.

Even more preferably, in the Nanobodies of the invention, at least twoof the CDR1, CDR2 and CDR3 sequences present are suitably chosen fromthe group consisting of the CDR1, CDR2 and CDR3 sequences, respectively,listed in Table A-1. Preferably, in this aspect, the remaining CDRsequence present is suitably chosen from the group of CDR sequences thathave at least 80%, preferably at least 90%, more preferably at least95%, even more preferably at least 99% sequence identity with at leastone of the corresponding CDR sequences listed in Table A-1; and/or fromthe group consisting of CDR sequences that have 3, 2 or only 1 aminoacid difference(s) with at least one of the corresponding sequenceslisted in Table A-1.

In particular, in the Nanobodies of the invention, at least the CDR3sequence is suitably chosen from the group consisting of the CDR3sequences listed in Table A-1, and either the CDR1 sequence or the CDR2sequence is suitably chosen from the group consisting of the CDR1 andCDR2 sequences, respectively, listed in Table A-1. Preferably, in thisaspect, the remaining CDR sequence present is suitably chosen from thegroup of CDR sequences that have at least 80%, preferably at least 90%,more preferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the corresponding CDR sequences listed inTable A-1; and/or from the group consisting of CDR sequences that have3, 2 or only 1 amino acid difference(s) with the corresponding CDRsequences listed in Table A-1.

Even more preferably, in the Nanobodies of the invention, all threeCDR1, CDR2 and CDR3 sequences present are suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed inTable A-1.

Also, generally, the combinations of CDR's listed in Table A-1 (i.e.those mentioned on the same line in Table A-1) are preferred. Thus, itis generally preferred that, when a CDR in a Nanobody of the inventionis a CDR sequence mentioned in Table A-1 or is suitably chosen from thegroup of CDR sequences that have at least 80%, preferably at least 90%,more preferably at least 95%, even more preferably at least 99% sequenceidentity with a CDR sequence listed in Table A-1; and/or from the groupconsisting of CDR sequences that have 3, 2 or only 1 amino aciddifference(s) with a CDR sequence listed in Table A-1, that at least oneand preferably both of the other CDR's are suitably chosen from the CDRsequences that belong to the same combination in Table A-1 (i.e.mentioned on the same line in Table A-1) or are suitably chosen from thegroup of CDR sequences that have at least 80%, preferably at least 90%,more preferably at least 95%, even more preferably at least 99% sequenceidentity with the CDR sequence(s) belonging to the same combinationand/or from the group consisting of CDR sequences that have 3, 2 or only1 amino acid difference(s) with the CDR sequence(s) belonging to thesame combination. The other preferences indicated in the aboveparagraphs also apply to the combinations of CDR's mentioned in TableA-1.

Thus, by means of non-limiting examples, a Nanobody of the invention canfor example comprise a CDR1 sequence that has more than 80% sequenceidentity with one of the CDR1 sequences mentioned in Table A-1, a CDR2sequence that has 3, 2 or 1 amino acid difference with one of the CDR2sequences mentioned in Table A-1 (but belonging to a differentcombination), and a CDR3 sequence.

Some preferred Nanobodies of the invention may for example comprise: (1)a CDR1 sequence that has more than 80% sequence identity with one of theCDR1 sequences mentioned in Table A-1; a CDR2 sequence that has 3, 2 or1 amino acid difference with one of the CDR2 sequences mentioned inTable A-1 (but belonging to a different combination); and a CDR3sequence that has more than 80% sequence identity with one of the CDR3sequences mentioned in Table A-1 (but belonging to a differentcombination); or (2) a CDR1 sequence that has more than 80% sequenceidentity with one of the CDR1 sequences mentioned in Table A-1; a CDR2sequence, and one of the CDR3 sequences listed in Table A-1; or (3) aCDR1 sequence; a CDR2 sequence that has more than 80% sequence identitywith one of the CDR2 sequence listed in Table A-1; and a CDR3 sequencethat has 3, 2 or 1 amino acid differences with the CDR3 sequencementioned in Table A-1 that belongs to the same combination as the CDR2sequence.

Some particularly preferred Nanobodies of the invention may for examplecomprise: (1) a CDR1 sequence that has more than 80% sequence identitywith one of the CDR1 sequences mentioned in Table A-1; a CDR2 sequencethat has 3, 2 or 1 amino acid difference with the CDR2 sequencementioned in Table A-1 that belongs to the same combination; and a CDR3sequence that has more than 80% sequence identity with the CDR3 sequencementioned in Table A-1 that belongs to the same combination; (2) a CDR1sequence; a CDR 2 listed in Table A-1 and a CDR3 sequence listed inTable A-1 (in which the CDR2 sequence and CDR3 sequence may belong todifferent combinations).

Some even more preferred Nanobodies of the invention may for examplecomprise: (1) a CDR1 sequence that has more than 80% sequence identitywith one of the CDR1 sequences mentioned in Table A-1; the CDR2 sequencelisted in Table A-1 that belongs to the same combination; and a CDR3sequence mentioned in Table A-1 that belongs to a different combination;or (2) a CDR1 sequence mentioned in Table A-1; a CDR2 sequence that has3, 2 or 1 amino acid differences with the CDR2 sequence mentioned inTable A-1 that belongs to the same combination; and a CDR3 sequence thathas more than 80% sequence identity with the CDR3 sequence listed inTable A-1 that belongs to the same or a different combination.

Particularly preferred Nanobodies of the invention may for examplecomprise a CDR1 sequence mentioned in Table A-1, a CDR2 sequence thathas more than 80% sequence identity with the CDR2 sequence mentioned inTable A-1 that belongs to the same combination; and the CDR3 sequencementioned in Table A-1 that belongs to the same combination.

In the most preferred Nanobodies of the invention, the CDR1, CDR2 andCDR3 sequences present are suitably chosen from one of the combinationsof CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1.

According to another preferred, but non-limiting aspect of the invention(a) CDR1 has a length of between 1 and 12 amino acid residues, andusually between 2 and 9 amino acid residues, such as 5, 6 or 7 aminoacid residues; and/or (b) CDR2 has a length of between 13 and 24 aminoacid residues, and usually between 15 and 21 amino acid residues, suchas 16 and 17 amino acid residues; and/or (c) CDR3 has a length ofbetween 2 and 35 amino acid residues, and usually between 3 and 30 aminoacid residues, such as between 6 and 23 amino acid residues.

In another preferred, but non-limiting aspect, the invention relates toa Nanobody in which the CDR sequences (as defined herein) have more than80%, preferably more than 90%, more preferably more than 95%, such as99% or more sequence identity (as defined herein) with the CDR sequencesof at least one of the amino acid sequences of SEQ ID NO's: 455 to 501,more preferably 455 to 457, 459, 460, 464 to 469.

Generally, Nanobodies with the above CDR sequences may be as furtherdescribed herein, and preferably have framework sequences that are alsoas further described herein. Thus, for example and as mentioned herein,such Nanobodies may be naturally occurring Nanobodies (from any suitablespecies), naturally occurring V_(HH) sequences (i.e. from a suitablespecies of Camelid) or synthetic or semi-synthetic amino acid sequencesor Nanobodies, including but not limited to partially humanizedNanobodies or V_(HH) sequences, fully humanized Nanobodies orV_(HH)-sequences, camelized heavy chain variable domain sequences, aswell as Nanobodies that have been obtained by the techniques mentionedherein.

Thus, in one specific, but non-limiting aspect, the invention relates toa humanized Nanobody, which consists of 4 framework regions (FR1 to FR4respectively) and 3 complementarity determining regions (CDR1 to CDR3respectively), in which CDR1 to CDR3 are as defined herein and in whichsaid humanized Nanobody comprises at least one humanizing substitution(as defined herein), and in particular at least one humanizingsubstitution in at least one of its framework sequences (as definedherein).

In another preferred, but non-limiting aspect, the invention relates toa Nanobody in which the CDR sequences have at least 70% amino acididentity, preferably at least 80% amino acid identity, more preferablyat least 90% amino acid identity, such as 95% amino acid identity ormore or even essentially 100% amino acid identity with the CDR sequencesof at least one of the amino acid sequences of SEQ ID NO's: 455 to 501,more preferably 455 to 457, 459, 460, 464 to 469. This degree of aminoacid identity can for example be determined by determining the degree ofamino acid identity (in a manner described herein) between said Nanobodyand one or more of the sequences of SEQ ID NO's: 455 to 501, morepreferably 455 to 457, 459, 460, 464 to 469, in which the amino acidresidues that form the framework regions are disregarded. SuchNanobodies can be as further described herein.

In another preferred, but non-limiting aspect, the invention relates toa Nanobody with an amino acid sequence that is chosen from the groupconsisting of SEQ ID NO's: 455 to 501, more preferably 455 to 457, 459,460, 464 to 469 or from the group consisting of from amino acidsequences that have more than 80%, preferably more than 90%, morepreferably more than 95%, such as 99% or more sequence identity (asdefined herein) with at least one of the amino acid sequences of SEQ IDNO's: 455 to 501, more preferably 455 to 457, 459, 460, 464 to 469.

Another preferred, but non-limiting aspect of the invention relates tohumanized variants of the Nanobodies of SEQ ID NO's: 455 to 501, morepreferably 455 to 457, 459, 460, 464 to 469, that comprise, compared tothe corresponding native V_(HH)-sequence, at least one humanizingsubstitution (as defined herein), and in particular at least onehumanizing substitution in at least one of its framework sequences (asdefined herein).

It will be clear to the skilled person that the Nanobodies that arementioned herein as “preferred” (or “more preferred”, “even morepreferred”, etc.) are also preferred (or more preferred, or even morepreferred, etc.) for use in the polypeptides described herein. Thus,polypeptides that comprise or essentially consist of one or more“preferred” Nanobodies of the invention will generally be preferred, andpolypeptides that comprise or essentially consist of one or more “morepreferred” Nanobodies of the invention will generally be more preferred,etc.

Generally, proteins or polypeptides that comprise or essentially consistof a single Nanobody (such as a single Nanobody of the invention) willbe referred to herein as “monovalent” proteins or polypeptides or as“monovalent constructs”, Proteins and polypeptides that comprise oressentially consist of two or more Nanobodies (such as at least twoNanobodies of the invention or at least one Nanobody of the inventionand at least one other Nanobody) will be referred to herein as“multivalent” proteins or polypeptides or as “multivalent constructs”,and these may provide certain advantages compared to the correspondingmonovalent Nanobodies of the invention. Some non-limiting examples ofsuch multivalent constructs will become clear from the furtherdescription herein.

According to one specific, but non-limiting aspect, a polypeptide of theinvention comprises or essentially consists of at least two Nanobodiesof the invention, such as two or three Nanobodies of the invention. Asfurther described herein, such multivalent constructs can providecertain advantages compared to a protein or polypeptide comprising oressentially consisting of a single Nanobody of the invention, such as amuch improved avidity for Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1,Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, more preferably Tie2,Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2 or Ang2. Suchmultivalent constructs will be clear to the skilled person based on thedisclosure herein.

According to another specific, but non-limiting aspect, a polypeptide ofthe invention comprises or essentially consists of at least one Nanobodyof the invention and at least one other binding unit (i.e. directedagainst another epitope, antigen, target, protein or polypeptide), whichis preferably also a Nanobody. Such proteins or polypeptides are alsoreferred to herein as “multispecific” proteins or polypeptides or as‘multispecific constructs”, and these may provide certain advantagescompared to the corresponding monovalent Nanobodies of the invention (aswill become clear from the further discussion herein of some preferred,but-nonlimiting multispecific constructs). Such multispecific constructswill be clear to the skilled person based on the disclosure herein.

According to yet another specific, but non-limiting aspect, apolypeptide of the invention comprises or essentially consists of atleast one Nanobody of the invention, optionally one or more furtherNanobodies, and at least one other amino acid sequence (such as aprotein or polypeptide) that confers at least one desired property tothe Nanobody of the invention and/or to the resulting fusion protein.Again, such fusion proteins may provide certain advantages compared tothe corresponding monovalent Nanobodies of the invention. Somenon-limiting examples of such amino acid sequences and of such fusionconstructs will become clear from the further description herein.

It is also possible to combine two or more of the above aspects, forexample to provide a trivalent bispecific construct comprising twoNanobodies of the invention and one other Nanobody, and optionally oneor more other amino acid sequences. Further non-limiting examples ofsuch constructs, as well as some constructs that are particularlypreferred within the context of the present invention, will become clearfrom the further description herein.

In the above constructs, the one or more Nanobodies and/or other aminoacid sequences may be directly linked to each other and/or suitablylinked to each other via one or more linker sequences. Some suitable butnon-limiting examples of such linkers will become clear from the furtherdescription herein.

In one specific aspect of the invention, a Nanobody of the invention ora compound, construct or polypeptide of the invention comprising atleast one Nanobody of the invention may have an increased half-life,compared to the corresponding amino acid sequence of the invention. Somepreferred, but non-limiting examples of such Nanobodies, compounds andpolypeptides will become clear to the skilled person based on thefurther disclosure herein, and for example comprise Nanobodies sequencesor polypeptides of the invention that have been chemically modified toincrease the half-life thereof (for example, by means of pegylation);amino acid sequences of the invention that comprise at least oneadditional binding site for binding to a serum protein (such as serumalbumin. Reference is for example made to the US provisional applicationby Ablynx N.V. entitled “Immunoglobulin domains with multiple bindingsites” filed on Nov. 27, 2006); or polypeptides of the invention thatcomprise at least one Nanobody of the invention that is linked to atleast one moiety (and in particular at least one amino acid sequence)that increases the half-life of the Nanobody of the invention. Examplesof polypeptides of the invention that comprise such half-life extendingmoieties or amino acid sequences will become clear to the skilled personbased on the further disclosure herein; and for example include, withoutlimitation, polypeptides in which the one or more Nanobodies of theinvention are suitable linked to one or more serum proteins or fragmentsthereof (such as serum albumin or suitable fragments thereof) or to oneor more binding units that can bind to serum proteins (such as, forexample, Nanobodies or (single) domain antibodies that can bind to serumproteins such as serum albumin, serum immunoglobulins such as IgG, ortransferrine); polypeptides in which a Nanobody of the invention islinked to an Fc portion (such as a human Fe) or a suitable part orfragment thereof; or polypeptides in which the one or more Nanobodies ofthe invention are suitable linked to one or more small proteins orpeptides that can bind to serum proteins (such as, without limitation,the proteins and peptides described in WO 91/01743, WO 01/45746, WO02/076489 and to the US provisional application of Ablynx N.V. entitled“Peptides capable of binding to serum proteins” of Ablynx. N.V. filed onDec. 5, 2006.

Again, as will be clear to the skilled person, such Nanobodies,compounds, constructs or polypeptides may contain one or more additionalgroups, residues, moieties or binding units, such as one or more furtheramino acid sequences and in particular one or more additional Nanobodies(i.e. not directed against Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1,Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, more preferably Tie2,Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2 or Ang2), so as toprovide a tri- of multispecific Nanobody construct.

Generally, the Nanobodies of the invention (or compounds, constructs orpolypeptides comprising the same) with increased half-life preferablyhave a half-life that is at least 1.5 times, preferably at least 2times, such as at least 5 times, for example at least 10 times or morethan 20 times, greater than the half-life of the corresponding aminoacid sequence of the invention per se. For example, the Nanobodies,compounds, constructs or polypeptides of the invention with increasedhalf-life may have a half-life that is increased with more than 1 hours,preferably more than 2 hours, more preferably more than 6 hours, such asmore than 12 hours, or even more than 24, 48 or 72 hours, compared tothe corresponding amino acid sequence of the invention per se.

In a preferred, but non-limiting aspect of the invention, suchNanobodies, compound, constructs or polypeptides of the inventionexhibit a serum half-life in human of at least about 12 hours,preferably at least 24 hours, more preferably at least 48 hours, evenmore preferably at least 72 hours or more. For example, compounds orpolypeptides of the invention may have a half-life of at least 5 days(such as about 5 to 10 days), preferably at least 9 days (such as about9 to 14 days), more preferably at least about 10 days (such as about 10to 15 days), or at least about 11 days (such as about 11 to 16 days),more preferably at least about 12 days (such as about 12 to 18 days ormore), or more than 14 days (such as about 14 to 19 days).

In another one aspect of the invention, a polypeptide of the inventioncomprises one or more (such as two or preferably one) Nanobodies of theinvention linked (optionally via one or more suitable linker sequences)to one or more (such as two and preferably one) amino acid sequencesthat allow the resulting polypeptide of the invention to cross the bloodbrain barrier. In particular, said one or more amino acid sequences thatallow the resulting polypeptides of the invention to cross the bloodbrain barrier may be one or more (such as two and preferably one)Nanobodies, such as the Nanobodies described in WO 02/057445, of whichFC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQ ID NO: 190 of WO06/040154) are preferred examples.

In particular, polypeptides comprising one or more Nanobodies of theinvention are preferably such that they:

-   -   bind to Tie1, Tie2, Ang1 Ang2, Ang3, Ang4, Angptl1, Angptl2,        Angptl3, Angptl4, Angptl5, or Angptl6, more preferably Tie2,        Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2 or Ang2 with        a dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or        less, and preferably 10⁻⁷ to 10⁻¹² moles/liter or less and more        preferably 10⁻⁸ to 10⁻¹² moles/liter (i.e. with an association        constant (K_(A)) of 10⁵ to 10¹² liter/moles or more, and        preferably 10⁷ to 10¹² liter/moles or more and more preferably        10⁸ to 10¹² liter/moles);        and/or such that they:    -   bind to Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,        Angptl3, Angptl4, Angptl5, or Angptl6, more preferably Tie2,        Ang2, Ang 1, Ang4, or Angptl4, more preferably Tie2 or Ang2 with        a k_(on)-rate of between 10²M⁻¹s⁻¹ to about 10⁷M⁻¹s⁻¹,        preferably between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more preferably        between 10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹        and 10⁷ M⁻¹s⁻¹;        and/or such that they:    -   bind to Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,        Angptl3, Angptl4, Angptl5, or Angptl6, more preferably Tie2,        Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2 or Ang2 with        a k_(off) rate between 1 s⁻¹ (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹        (providing a near irreversible complex with a t_(1/2) of        multiple days), preferably between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more        preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as between        10⁻⁴s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, a polypeptide that contains only one amino acid sequence ofthe invention is preferably such that it will bind to Tie1, Tie2, Ang1,Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, morepreferably Tie2 or Ang2 with an affinity less than 500 nM, preferablyless than 200 nM, more preferably less than 10 nM, such as less than 500μM. In this respect, it will be clear to the skilled person that apolypeptide that contains two or more Nanobodies of the invention maybind to Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3,Angptl4, Angptl5, or Angptl6, more preferably Tie2, Ang2, Ang1, Ang4, orAngptl4, more preferably Tie2 or Ang2 with an increased avidity,compared to a polypeptide that contains only one amino acid sequence ofthe invention.

Some preferred IC₅₀ values for binding of the amino acid sequences orpolypeptides of the invention to Tie1, Tie2, Ang1, Ang2, Ang3, Ang4,Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, more preferablyTie2, Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2 or Ang2 willbecome clear from the further description and examples herein.

Other polypeptides according to this preferred aspect of the inventionmay for example be chosen from the group consisting of amino acidsequences that have more than 80%, preferably more than 90%, morepreferably more than 95%, such as 99% or more “sequence identity” (asdefined herein) with one or more of the amino acid sequences of SEQ IDNO's: 455 to 501, more preferably, 455 to 457, 459, 460, 464 to 469, inwhich the Nanobodies comprised within said amino acid sequences arepreferably as further defined herein.

Another aspect of this invention relates to a nucleic acid that encodesa Nanobody of the invention or a polypeptide of the invention comprisingthe same. Again, as generally described herein for the nucleic acids ofthe invention, such a nucleic acid may be in the form of a geneticconstruct, as defined herein.

In another aspect, the invention relates to host or host cell thatexpresses or that is capable of expressing a Nanobody of the inventionand/or a polypeptide of the invention comprising the same; and/or thatcontains a nucleic acid of the invention. Some preferred butnon-limiting examples of such hosts or host cells will become clear fromthe further description herein.

Another aspect of the invention relates to a product or compositioncontaining or comprising at least one Nanobody of the invention, atleast one polypeptide of the invention and/or at least one nucleic acidof the invention, and optionally one or more further components of suchcompositions known per se, i.e. depending on the intended use of thecomposition. Such a product or composition may for example be apharmaceutical composition (as described herein), a veterinarycomposition or a product or composition for diagnostic use (as alsodescribed herein). Some preferred but non-limiting examples of suchproducts or compositions will become clear from the further descriptionherein.

The invention further relates to methods for preparing or generating theNanobodies, polypeptides, nucleic acids, host cells, products andcompositions described herein. Some preferred but non-limiting examplesof such methods will become clear from the further description herein.

The invention further relates to applications and uses of theNanobodies, polypeptides, nucleic acids, host cells, products andcompositions described herein, as well as to methods for the preventionand/or treatment for diseases and disorders associated with Tie1, Tie2,Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, morepreferably Tie2 or Ang2. Some preferred but non-limiting applicationsand uses will become clear from the further description herein.

Other aspects, embodiments, advantages and applications of the inventionwill also become clear from the further description herein below.

Generally, it should be noted that the term Nanobody as used herein inits broadest sense is not limited to a specific biological source or toa specific method of preparation. For example, as will be discussed inmore detail below, the Nanobodies of the invention can generally beobtained: (1) by isolating the V_(HH) domain of a naturally occurringheavy chain antibody; (2) by expression of a nucleotide sequenceencoding a naturally occurring V_(HH) domain; (3) by “humanization” (asdescribed herein) of a naturally occurring V_(HH) domain or byexpression of a nucleic acid encoding a such humanized V_(HH) domain;(4) by “camelization” (as described herein) of a naturally occurringV_(H) domain from any animal species, and in particular a from speciesof mammal, such as from a human being, or by expression of a nucleicacid encoding such a camelized V_(H) domain; (5) by “camelization” of a“domain antibody” or “Dab” as described by Ward et al (supra), or byexpression of a nucleic acid encoding such a camelized V_(H) domain; (6)by using synthetic or semi-synthetic techniques for preparing proteins,polypeptides or other amino acid sequences known per se; (7) bypreparing a nucleic acid encoding a Nanobody using techniques fornucleic acid synthesis known per se, followed by expression of thenucleic acid thus obtained; and/or (8) by any combination of one or moreof the foregoing. Suitable methods and techniques for performing theforegoing will be clear to the skilled person based on the disclosureherein and for example include the methods and techniques described inmore detail herein.

One preferred class of Nanobodies corresponds to the V_(HH) domains ofnaturally occurring heavy chain antibodies directed against Tie1, Tie2,Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, morepreferably Tie2 or Ang2. As further described herein, such V_(HH)sequences can generally be generated or obtained by suitably immunizinga species of Camelid with Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1,Angptl2, Angptl3, Angptl4, Angptl5, or Angptl4, more preferably Tie2,Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2 or Ang2 (i.e. so asto raise an immune response and/or heavy chain antibodies directedagainst Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3,Angptl4, Angptl5, or Angptl6, more preferably Tie2, Ang2, Ang 1, Ang4,or Angptl4, more preferably Tie2 or Ang2), by obtaining a suitablebiological sample from said Camelid (such as a blood sample, serumsample or sample of B-cells), and by generating V_(HH) sequencesdirected against Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,Angptl3, Angptl4, Angptl5, or Angptl6, more preferably Tie2, Ang2, Ang1,Ang4, or Angptl4, more preferably Tie2 or Ang2, starting from saidsample, using any suitable technique known per se. Such techniques willbe clear to the skilled person and/or are further described herein.

Alternatively, such naturally occurring V_(HH) domains against Tie1,Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4,Angptl5, or Angptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4,more preferably Tie2 or Ang2, can be obtained from naïve libraries ofCamelid V_(HH) sequences, for example by screening such a library usingTie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4,Angptl5, or Angptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4,more preferably Tie2 or Ang2, or at least one part, fragment, antigenicdeterminant or epitope thereof using one or more screening techniquesknown per se. Such libraries and techniques are for example described inWO 99/37681, WO 01/90190, WO 03/025020 and WO 03/035694. Alternatively,improved synthetic or semi-synthetic libraries derived from naïve V_(HH)libraries may be used, such as libraries obtained from naïve V_(HH)libraries by techniques such as random mutagenesis and/or CDR shuffling,as for example described in WO 00/43507.

Thus, in another aspect, the invention relates to a method forgenerating Nanobodies, that are directed against Tie1, Tie2, Ang1, Ang2,Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl5,more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2or Ang2. In one aspect, said method at least comprises the steps of:

-   a) providing a set, collection or library of Nanobody sequences; and-   b) screening said set, collection or library of Nanobody sequences    for Nanobody sequences that can bind to and/or have affinity for    Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3,    Angptl4, Angptl5, or Angptl6, more preferably Tie2, Ang2, Ang1,    Ang4, or Angptl4, more preferably Tie2 or Ang2;    and-   c) isolating the amino acid sequence(s) that can bind to and/or have    affinity for Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,    Angptl3, Angptl4, Angptl5, or Angptl6, more preferably Tie2, Ang2,    Ang1, Ang4, or Angptl4, more preferably Tie2 or Ang2. In such a    method, the set, collection or library of Nanobody sequences may be    a naïve set, collection or library of Nanobody sequences; a    synthetic or semi-synthetic set, collection or library of Nanobody    sequences; and/or a set, collection or library of Nanobody sequences    that have been subjected to affinity maturation.

In a preferred aspect of this method, the set, collection or library ofNanobody sequences may be an immune set, collection or library ofNanobody sequences, and in particular an immune set, collection orlibrary of V_(HH) sequences, that have been derived from a species ofCamelid that has been suitably immunized with Tie1, Tie2, Ang1, Ang2,Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6,more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2or Ang2 or with a suitable antigenic determinant based thereon orderived there from, such as an antigenic part, fragment, region, domain,loop or other epitope thereof. In one particular aspect, said antigenicdeterminant may be an extracellular part, region, domain, loop or otherextracellular epitope(s).

In the above methods, the set, collection or library of Nanobody orV_(HH) sequences may be displayed on a phage, phagemid, ribosome orsuitable micro-organism (such as yeast), such as to facilitatescreening. Suitable methods, techniques and host organisms fordisplaying and screening (a set, collection or library of) Nanobodysequences will be clear to the person skilled in the art, for example onthe basis of the further disclosure herein. Reference is also made to WO03/054016 and to the review by Hoogenboom in Nature Biotechnology, 23,9, 1105-1116 (2005).

In another aspect, the method for generating Nanobody sequencescomprises at least the steps of:

-   a) providing a collection or sample of cells derived from a species    of Camelid that express immunoglobulin sequences;-   b) screening said collection or sample of cells for (i) cells that    express an immunoglobulin sequence that can bind to and/or have    affinity for Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,    Angptl3, Angptl4, Angptl5, or Angptl6, more preferably Tie2, Ang2,    Ang1, Ang4, or Angptl4, more preferably Tie2 or Ang2; and (ii) cells    that express heavy chain antibodies, in which sub steps (i) and (ii)    can be performed essentially as a single screening step or in any    suitable order as two separate screening steps, so as to provide at    least one cell that expresses a heavy chain antibody that can bind    to and/or has affinity for Tie1, Tie2, Ang1, Ang2, Ang3, Ang4,    Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, more    preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2    or Ang2;    and-   c) either (i) isolating from said cell the V_(HH) sequence present    in said heavy chain antibody; or (ii) isolating from said cell a    nucleic acid sequence that encodes the V_(HH) sequence present in    said heavy chain antibody, followed by expressing said V_(HH)    domain.

In the method according to this aspect, the collection or sample ofcells may for example be a collection or sample of B-cells. Also, inthis method, the sample of cells may be derived from a Camelid that hasbeen suitably immunized with Tie1, Tie2, Ang1, Ang2, Ang3, Ang4,Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, more preferablyTie2, Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2 or Ang2 or asuitable antigenic determinant based thereon or derived there from, suchas an antigenic part, fragment, region, domain, loop or other epitopethereof. In one particular aspect, said antigenic determinant may be anextracellular part, region, domain, loop or other extracellularepitope(s).

The above method may be performed in any suitable manner, as will beclear to the skilled person. Reference is for example made to EP 0 542810, WO 05/19824, WO 04/051268 and WO 04/106377. The screening of stepb) is preferably performed using a flow cytometry technique such asFACS. For this, reference is for example made to Lieby et al., Blood,Vol. 97, No. 12, 3820. Particular reference is made to the so-called“Nanoclone™” technique described in International application WO06/079372 by Ablynx N.V.

In another aspect, the method for generating an amino acid sequencedirected against Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,Angptl3, Angptl4, Angptl5, or Angptl6, more preferably Tie2, Ang2, Ang1,Ang4, or Angptl4, more preferably Tie2 or Ang2 may comprise at least thesteps of:

-   a) providing a set, collection or library of nucleic acid sequences    encoding heavy chain antibodies or Nanobody sequences;-   b) screening said set, collection or library of nucleic acid    sequences for nucleic acid sequences that encode a heavy chain    antibody or a Nanobody sequence that can bind to and/or has affinity    for Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3,    Angptl4, Angptl5, or Angptl6, more preferably Tie2, Ang2, Ang1,    Ang4, or Angptl4, more preferably Tie2 or Ang2;    and-   c) isolating said nucleic acid sequence, followed by expressing the    V_(HH) sequence present in said heavy chain antibody or by    expressing said Nanobody sequence, respectively.

In such a method, the set, collection or library of nucleic acidsequences encoding heavy chain antibodies or Nanobody sequences may forexample be a set, collection or library of nucleic acid sequencesencoding a naïve set, collection or library of heavy chain antibodies orV_(HH) sequences; a set, collection or library of nucleic acid sequencesencoding a synthetic or semi-synthetic set, collection or library ofNanobody sequences; and/or a set, collection or library of nucleic acidsequences encoding a set, collection or library of Nanobody sequencesthat have been subjected to affinity maturation.

In a preferred aspect of this method, the set, collection or library ofamino acid sequences may be an immune set, collection or library ofnucleic acid sequences encoding heavy chain antibodies or V_(HH)sequences derived from a Camelid that has been suitably immunized withTie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4,Angptl5, or Angptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4,more preferably Tie2 or Ang2 or with a suitable antigenic determinantbased thereon or derived there from, such as an antigenic part,fragment, region, domain, loop or other epitope thereof. In oneparticular aspect, said antigenic determinant may be an extracellularpart, region, domain, loop or other extracellular epitope(s).

In the above methods, the set, collection or library of nucleotidesequences may be displayed on a phage, phagemid, ribosome or suitablemicro-organism (such as yeast), such as to facilitate screening.Suitable methods, techniques and host organisms for displaying andscreening (a set, collection or library of) nucleotide sequencesencoding amino acid sequences will be clear to the person skilled in theart, for example on the basis of the further disclosure herein.Reference is also made to WO 03/054016 and to the review by Hoogenboomin Nature Biotechnology, 23, 9, 1105-1116 (2005).

As will be clear to the skilled person, the screening step of themethods described herein can also be performed as a selection step.Accordingly the term “screening” as used in the present description cancomprise selection, screening or any suitable combination of selectionand/or screening techniques. Also, when a set, collection or library ofsequences is used, it may contain any suitable number of sequences, suchas 1, 2, 3 or about 5, 10, 50, 100, 500, 1000, 5000, 10⁴, 10⁵, 10⁶, 10⁷,10⁸ or more sequences.

Also, one or more or all of the sequences in the above set, collectionor library of amino acid sequences may be obtained or defined byrational, or semi-empirical approaches such as computer modellingtechniques or biostatics or data mining techniques.

Furthermore, such a set, collection or library can comprise one, two ormore sequences that are variants from one another (e.g. with designedpoint mutations or with randomized positions), compromise multiplesequences derived from a diverse set of naturally diversified sequences(e.g. an immune library)), or any other source of diverse sequences (asdescribed for example in Hoogenboom et al, Nat Biotechnol 23:1105, 2005and Binz et al, Nat Biotechnol 2005, 23:1247). Such set, collection orlibrary of sequences can be displayed on the surface of a phageparticle, a ribosome, a bacterium, a yeast cell, a mammalian cell, andlinked to the nucleotide sequence encoding the amino acid sequencewithin these carriers. This makes such set, collection or libraryamenable to selection procedures to isolate the desired amino acidsequences of the invention. More generally, when a sequence is displayedon a suitable host or host cell, it is also possible (and customary) tofirst isolate from said host or host cell a nucleotide sequence thatencodes the desired sequence, and then to obtain the desired sequence bysuitably expressing said nucleotide sequence in a suitable hostorganism. Again, this can be performed in any suitable manner known perse, as will be clear to the skilled person.

Yet another technique for obtaining V_(HH) sequences or Nanobodysequences directed against Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1,Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, more preferably Tie2,Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2 or Ang2 involvessuitably immunizing a transgenic mammal that is capable of expressingheavy chain antibodies (i.e. so as to raise an immune response and/orheavy chain antibodies directed against Tie1, Tie2, Ang1, Ang2, Ang3,Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, morepreferably Tie2, Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2 orAng2), obtaining a suitable biological sample from said transgenicmammal that contains (nucleic acid sequences encoding) said V_(HH)sequences or Nanobody sequences (such as a blood sample, serum sample orsample of B-cells), and then generating V_(HH) sequences directedagainst Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3,Angptl4, Angptl5, or Angptl6, more preferably Tie2, Ang2, Ang1, Ang4, orAngptl4, more preferably Tie2 or Ang2, starting from said sample, usingany suitable technique known per se (such as any of the methodsdescribed herein or a hybridoma technique). For example, for thispurpose, the heavy chain antibody-expressing mice and the furthermethods and techniques described in WO 02/085945, WO 04/049794 and WO06/008548 and Janssens et al., Proc. Natl. Acad. Sci. USA. 2006 Oct. 10;103(41):15130-5 can be used. For example, such heavy chain antibodyexpressing mice can express heavy chain antibodies with any suitable(single) variable domain, such as (single) variable domains from naturalsources (e.g. human (single) variable domains, Camelid (single) variabledomains or shark (single) variable domains), as well as for examplesynthetic or semi-synthetic (single) variable domains.

The invention also relates to the V_(HH) sequences or Nanobody sequencesthat are obtained by the above methods, or alternatively by a methodthat comprises the one of the above methods and in addition at least thesteps of determining the nucleotide sequence or amino acid sequence ofsaid V_(HH) sequence or Nanobody sequence; and of expressing orsynthesizing said V_(HH) sequence or Nanobody sequence in a manner knownper se, such as by expression in a suitable host cell or host organismor by chemical synthesis.

As mentioned herein, a particularly preferred class of Nanobodies of theinvention comprises Nanobodies with an amino acid sequence thatcorresponds to the amino acid sequence of a naturally occurring V_(HH)domain, but that has been “humanized”, i.e. by replacing one or moreamino acid residues in the amino acid sequence of said naturallyoccurring V_(HH) sequence (and in particular in the framework sequences)by one or more of the amino acid residues that occur at thecorresponding position(s) in a V_(H) domain from a conventional 4-chainantibody from a human being (e.g. indicated above). This can beperformed in a manner known per se, which will be clear to the skilledperson, for example on the basis of the further description herein andthe prior art on humanization referred to herein. Again, it should benoted that such humanized Nanobodies of the invention can be obtained inany suitable manner known per se (i.e. as indicated under points (1)-(8)above) and thus are not strictly limited to polypeptides that have beenobtained using a polypeptide that comprises a naturally occurring V_(HH)domain as a starting material.

Another particularly preferred class of Nanobodies of the inventioncomprises Nanobodies with an amino acid sequence that corresponds to theamino acid sequence of a naturally occurring V_(H) domain, but that hasbeen “camelized”, i.e. by replacing one or more amino acid residues inthe amino acid sequence of a naturally occurring V_(H) domain from aconventional 4-chain antibody by one or more of the amino acid residuesthat occur at the corresponding position(s) in a V_(HH) domain of aheavy chain antibody. This can be performed in a manner known per se,which will be clear to the skilled person, for example on the basis ofthe further description herein. Such “camelizing” substitutions arepreferably inserted at amino acid positions that form and/or are presentat the V_(H)-V_(L) interface, and/or at the so-called Camelidae hallmarkresidues, as defined herein (see for example WO 94/04678 and Davies andRiechmann (1994 and 1996), supra). Preferably, the V_(H) sequence thatis used as a starting material or starting point for generating ordesigning the camelized Nanobody is preferably a V_(H) sequence from amammal, more preferably the V_(H) sequence of a human being, such as aV_(H)3 sequence. However, it should be noted that such camelizedNanobodies of the invention can be obtained in any suitable manner knownper se (i.e. as indicated under points (1)-(8) above) and thus are notstrictly limited to polypeptides that have been obtained using apolypeptide that comprises a naturally occurring V_(H) domain as astarting material.

For example, again as further described herein, both “humanization” and“camelization” can be performed by providing a nucleotide sequence thatencodes a naturally occurring V_(HH) domain or V_(H) domain,respectively, and then changing, in a manner known per se, one or morecodons in said nucleotide sequence in such a way that the new nucleotidesequence encodes a “humanized” or “camelized” Nanobody of the invention,respectively. This nucleic acid can then be expressed in a manner knownper se, so as to provide the desired Nanobody of the invention.Alternatively, based on the amino acid sequence of a naturally occurringV_(HH) domain or V_(H) domain, respectively, the amino acid sequence ofthe desired humanized or camelized Nanobody of the invention,respectively, can be designed and then synthesized de novo usingtechniques for peptide synthesis known per se. Also, based on the aminoacid sequence or nucleotide sequence of a naturally occurring V_(HH)domain or V_(H) domain, respectively, a nucleotide sequence encoding thedesired humanized or camelized Nanobody of the invention, respectively,can be designed and then synthesized de novo using techniques fornucleic acid synthesis known per se, after which the nucleic acid thusobtained can be expressed in a manner known per se, so as to provide thedesired Nanobody of the invention.

Other suitable methods and techniques for obtaining the Nanobodies ofthe invention and/or nucleic acids encoding the same, starting fromnaturally occurring V_(H) sequences or preferably V_(HH) sequences, willbe clear from the skilled person, and may for example comprise combiningone or more parts of one or more naturally occurring V_(H) sequences(such as one or more FR sequences and/or CDR sequences), one or moreparts of one or more naturally occurring V_(HH) sequences (such as oneor more FR sequences or CDR sequences), and/or one or more synthetic orsemi-synthetic sequences, in a suitable manner, so as to provide aNanobody of the invention or a nucleotide sequence or nucleic acidencoding the same (which may then be suitably expressed). Nucleotidesequences encoding framework sequences of V_(HH) sequences or Nanobodieswill be clear to the skilled person based on the disclosure hereinand/or the further prior art cited herein (and/or may alternatively beobtained by PCR starting from the nucleotide sequences obtained usingthe methods described herein) and may be suitably combined withnucleotide sequences that encode the desired CDR's (for example, by PCRassembly using overlapping primers), so as to provide a nucleic acidencoding a Nanobody of the invention.

As mentioned herein, Nanobodies may in particular be characterized bythe presence of one or more “Hallmark residues” (as described herein) inone or more of the framework sequences.

Thus, according to one preferred, but non-limiting aspect of theinvention, a Nanobody in its broadest sense can be generally defined asa polypeptide comprising:

-   a) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 108    according to the Kabat numbering is Q;    and/or:-   b) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 45    according to the Kabat numbering is a charged amino acid (as defined    herein) or a cysteine residue, and position 44 is preferably an E;    and/or:-   c) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 103    according to the Kabat numbering is chosen from the group consisting    of P, R and S, and is in particular chosen from the group consisting    of R and S.

Thus, in a first preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which

-   a) the amino acid residue at position 108 according to the Kabat    numbering is Q; and/or in which:-   b) the amino acid residue at position 45 according to the Kabat    numbering is a charged amino acid or a cysteine and the amino acid    residue at position 44 according to the Kabat numbering is    preferably E;    and/or in which:-   c) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S, and is    in particular chosen from the group consisting of R and S;    and in which:-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In particular, a Nanobody in its broadest sense can be generally definedas a polypeptide comprising:

-   a) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 108    according to the Kabat numbering is Q;    and/or:-   b) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 44    according to the Kabat numbering is E and in which the amino acid    residue at position 45 according to the Kabat numbering is an R;    and/or:-   c) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarily determining    regions/sequences, in which the amino acid residue at position 103    according to the Kabat numbering is chosen from the group consisting    of P, R and S, and is in particular chosen from the group consisting    of R and S.

Thus, according to a preferred, but non-limiting aspect, a Nanobody ofthe invention may have the structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which

-   a) the amino acid residue at position 108 according to the Kabat    numbering is Q; and/or in which:-   b) the amino acid residue at position 44 according to the Kabat    numbering is E and in which the amino acid residue at position 45    according to the Kabat numbering is an R;    and/or in which:-   c) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S, and is    in particular chosen from the group consisting of R and S;    and in which:-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In particular, a Nanobody against Tie1, Tie2, Ang1, Ang2, Ang3, Ang4,Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, more preferablyTie2, Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2 or Ang2according to the invention may have the structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which

-   a) the amino acid residue at position 108 according to the Kabat    numbering is Q;    and/or in which:-   b) the amino acid residue at position 44 according to the Kabat    numbering is E and in which the amino acid residue at position 45    according to the Kabat numbering is an R;    and/or in which:-   c) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S, and is    in particular chosen from the group consisting of R and S;    and in which:-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In particular, according to one preferred, but non-limiting aspect ofthe invention, a Nanobody can generally be defined as a polypeptidecomprising an amino acid sequence that is comprised of four frameworkregions/sequences interrupted by three complementarity determiningregions/sequences, in which;

-   a-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of A, G, E, D, G, Q,    R, S, L; and is preferably chosen from the group consisting of G, E    or Q; and-   a-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R or C; and is    preferably chosen from the group consisting of L or R; and-   a-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R or S; and is    preferably W or R, and is most preferably W;-   a-4) the amino acid residue at position 108 according to the Kabat    numbering is Q;    or in which:-   b-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of E and Q; and-   b-2) the amino acid residue at position 45 according to the Kabat    numbering is R; and-   b-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R and S; and is    preferably W;-   b-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; and is    preferably Q;    or in which:-   c-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of A, G, E, D, Q, R, S    and L; and is preferably chosen from the group consisting of G, E    and Q; and-   c-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R and C; and is    preferably chosen from the group consisting of L and R; and-   c-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S; and is    in particular chosen from the group consisting of R and S; and-   c-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; is    preferably Q;    and in which-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   a-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of A, G, E, D, G, Q,    R, S, L; and is preferably chosen from the group consisting of G, E    or Q;    and in which:-   a-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R or C; and is    preferably chosen from the group consisting of L or R;    and in which:-   a-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R or S; and is    preferably W or R, and is most preferably W; and in which-   a-4) the amino acid residue at position 108 according to the Kabat    numbering is Q;    and in which:-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   b-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of E and Q;    and in which:-   b-2) the amino acid residue at position 45 according to the Kabat    numbering is R; and in which:-   b-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R and S; and is    preferably W;    and in which:-   b-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; and is    preferably Q;    and in which:-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   c-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of A, G, E, D, Q, R, S    and L; and is preferably chosen from the group consisting of G, E    and Q;    and in which:-   c-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R and C; and is    preferably chosen from the group consisting of L and R;    and in which:-   c-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S; and is    in particular chosen from the group consisting of R and S;    and in which:-   c-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; is    preferably Q;    and in which:-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

Two particularly preferred, but non-limiting groups of the Nanobodies ofthe invention are those according to a) above; according to (a-1) to(a-4) above; according to b) above; according to (b-1) to (b-4) above;according to (c) above; and/or according to (c-1) to (c-4) above, inwhich either:

-   i) the amino acid residues at positions 44-47 according to the Kabat    numbering form the sequence GLEW (or a GLEW-like sequence as    described herein) and the amino acid residue at position 108 is Q;    or in which:-   ii) the amino acid residues at positions 43-46 according to the    Kabat numbering form the sequence KERE or KQRE (or a KERE-like    sequence as described) and the amino acid residue at position 108 is    Q or L, and is preferably Q.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   i) the amino acid residues at positions 44-47 according to the Kabat    numbering form the sequence GLEW (or a GLEW-like sequence as defined    herein) and the amino acid residue at position 108 is Q;    and in which:-   ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   i) the amino acid residues at positions 43-46 according to the Kabat    numbering form the sequence KERE or KQRE (or a KERE-like sequence)    and the amino acid residue at position 108 is Q or L, and is    preferably Q;    and in which:-   ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In the Nanobodies of the invention in which the amino acid residues atpositions 43-46 according to the Kabat numbering form the sequence KEREor KQRE, the amino acid residue at position 37 is most preferably F. Inthe Nanobodies of the invention in which the amino acid residues atpositions 44-47 according to the Kabat numbering form the sequence GLEW,the amino acid residue at position 37 is chosen from the groupconsisting of Y, H, L, V or F, and is most preferably V.

Thus, without being limited hereto in any way, on the basis of the aminoacid residues present on the positions mentioned above, the Nanobodiesof the invention can generally be classified on the basis of thefollowing three groups:

-   i) The “GLEW-group”: Nanobodies with the amino acid sequence GLEW at    positions 44-47 according to the Kabat numbering and Q at position    108 according to the Kabat numbering. As further described herein,    Nanobodies within this group usually have a V at position 37, and    can have a W, P, R or S at position 103, and preferably have a W at    position 103. The GLEW group also comprises some GLEW-like sequences    such as those mentioned in Table A-3 below. More generally, and    without limitation, Nanobodies belonging to the GLEW-group can be    defined as Nanobodies with a G at position 44 and/or with a W at    position 47, in which position 46 is usually E and in which    preferably position 45 is not a charged amino acid residue and not    cysteine;-   ii) The “KERE-group”: Nanobodies with the amino acid sequence KERB    or KQRE (or another KERE-like sequence) at positions 43-46 according    to the Kabat numbering and Q or L at position 108 according to the    Kabat numbering. As further described herein, Nanobodies within this    group usually have a F at position 37, an L or F at position 47; and    can have a W, P, R or S at position 103, and preferably have a W at    position 103. More generally, and without limitation, Nanobodies    belonging to the KERE-group can be defined as Nanobodies with a K, Q    or R at position 44 (usually K) in which position 45 is a charged    amino acid residue or cysteine, and position 47 is as further    defined herein;-   iii) The “103 P, R, S-group”: Nanobodies with a P, R or S at    position 103. These Nanobodies can have either the amino acid    sequence GLEW at positions 44-47 according to the Kabat numbering or    the amino acid sequence KERE or KQRE at positions 43-46 according to    the Kabat numbering, the latter most preferably in combination with    an F at position 37 and an L or an F at position 47 (as defined for    the KERE-group); and can have Q or L at position 108 according to    the Kabat numbering, and preferably have Q.

Also, where appropriate, Nanobodies may belong to (i.e. havecharacteristics of) two or more of these classes. For example, onespecifically preferred group of Nanobodies has GLEW or a GLEW-likesequence at positions 44-47; P, R or S (and in particular R) at position103; and Q at position 108 (which may be humanized to L).

More generally, it should be noted that the definitions referred toabove describe and apply to Nanobodies in the form of a native (i.e.non-humanized) V_(HH) sequence, and that humanized variants of theseNanobodies may contain other amino acid residues than those indicatedabove (i.e. one or more humanizing substitutions as defined herein). Forexample, and without limitation, in some humanized Nanobodies of theGLEW-group or the 103 P, R, S-group, Q at position 108 may be humanizedto 108L. As already mentioned herein, other humanizing substitutions(and suitable combinations thereof) will become clear to the skilledperson based on the disclosure herein. In addition, or alternatively,other potentially useful humanizing substitutions can be ascertained bycomparing the sequence of the framework regions of a naturally occurringV_(HH) sequence with the corresponding framework sequence of one or moreclosely related human V_(H) sequences, after which one or more of thepotentially useful humanizing substitutions (or combinations thereof)thus determined can be introduced into said V_(HH) sequence (in anymanner known per se, as further described herein) and the resultinghumanized V_(HH) sequences can be tested for affinity for the target,for stability, for ease and level of expression, and/or for otherdesired properties. In this way, by means of a limited degree of trialand error, other suitable humanizing substitutions (or suitablecombinations thereof) can be determined by the skilled person based onthe disclosure herein. Also, based on the foregoing, (the frameworkregions of) a Nanobody may be partially humanized or fully humanized.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may be a Nanobody belonging to the GLEW-group (as definedherein), and in which CDR1, CDR2 and CDR3 are as defined herein, and arepreferably as defined according to one of the preferred aspects herein,and are more preferably as defined according to one of the morepreferred aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may be a Nanobody belonging to the KERE-group (as definedherein), and CDR1, CDR2 and CDR3 are as defined herein, and arepreferably as defined according to one of the preferred aspects herein,and are more preferably as defined according to one of the morepreferred aspects herein.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may be a Nanobody belonging to the 103 P, R, S-group (asdefined herein), and in which CDR1, CDR2 and CDR3 are as defined herein,and are preferably as defined according to one of the preferred aspectsherein, and are more preferably as defined according to one of the morepreferred aspects herein.

Also, more generally and in addition to the 108Q, 43E/44R and 103 P,R,Sresidues mentioned above, the Nanobodies of the invention can contain,at one or more positions that in a conventional V_(H) domain would form(part of) the V_(H)/V_(L) interface, one or more amino acid residuesthat are more highly charged than the amino acid residues that naturallyoccur at the same position(s) in the corresponding naturally occurringV_(H) sequence, and in particular one or more charged amino acidresidues (as mentioned in Table A-2). Such substitutions include, butare not limited to, the GLEW-like sequences mentioned in Table A-3below; as well as the substitutions that are described in theInternational Application WO 00/29004 for so-called “microbodies”, e.g.so as to obtain a Nanobody with Q at position 108 in combination withKLEW at positions 44-47. Other possible substitutions at these positionswill be clear to the skilled person based upon the disclosure herein.

In one aspect of the Nanobodies of the invention, the amino acid residueat position 83 is chosen from the group consisting of L, M, S, V and W;and is preferably L. Also, in one aspect of the Nanobodies of theinvention, the amino acid residue at position 83 is chosen from thegroup consisting of R, K, N, E, G, I, T and Q; and is most preferablyeither K or E (for Nanobodies corresponding to naturally occurringV_(HH) domains) or R (for “humanized” Nanobodies, as described herein).The amino acid residue at position 84 is chosen from the groupconsisting of P, A, R, S, D T, and V in one aspect, and is mostpreferably P (for Nanobodies corresponding to naturally occurring V_(HH)domains) or R (for “humanized” Nanobodies, as described herein).

Furthermore, in one aspect of the Nanobodies of the invention, the aminoacid residue at position 104 is chosen from the group consisting of Gand D; and is most preferably G.

Collectively, the amino acid residues at positions 11, 37, 44, 45, 47,83, 84, 103, 104 and 108, which in the Nanobodies are as mentionedabove, will also be referred to herein as the “Hallmark Residues”. TheHallmark Residues and the amino acid residues at the correspondingpositions of the most closely related human V_(H) domain, V_(H)3, aresummarized in Table A-3.

Some especially preferred but non-limiting combinations of theseHallmark Residues as occur in naturally occurring V_(HH) domains arementioned in Table A-4. For comparison, the corresponding amino acidresidues of the human V_(H)3 called DP-47 have been indicated initalics.

TABLE A-3 Hallmark Residues in Nanobodies Position Human V_(H)3 HallmarkResidues 11 L, V; predominantly L, M, S, V, W; preferably L L 37 V, I,F; usually V F⁽¹⁾, Y, H, I, L or V, preferably F⁽¹⁾ or Y    44⁽⁸⁾ GG⁽²⁾, E⁽³⁾, A, D, Q, R, S, L; preferably G⁽²⁾, E⁽³⁾ or Q; mostpreferably G⁽²⁾ or E⁽³⁾    45⁽⁸⁾ L L⁽²⁾, R⁽³⁾, C, I, L, P, Q, V;preferably L⁽²⁾ or R⁽³⁾    47⁽⁸⁾ W, Y W⁽²⁾, L⁽¹⁾ or F⁽¹⁾, A, G, I, M, R,S, V or Y; preferably W⁽²⁾, L⁽¹⁾, F⁽¹⁾ or R 83 R or K; usually R R,K⁽⁵⁾, N, E⁽⁵⁾, G, I, M, Q or T; preferably K or R; most preferably K 84A, T, D; P⁽⁵⁾, A, L, R, S, T, D, V; preferably P predominantly A 103  WW⁽⁴⁾, P⁽⁶⁾, R⁽⁶⁾, S; preferably W 104  G G or D; preferably G 108  L, Mor T; Q, L⁽⁷⁾ or R; preferably Q or L⁽⁷⁾ predominantly L Notes: ⁽¹⁾Inparticular, but not exclusively, in combination with KERE or KQRE atpositions 43-46. ⁽²⁾Usually as GLEW at positions 44-47. ⁽³⁾Usually asKERE or KQRE at positions 43-46, e.g. as KEREL, KEREF, KQREL, KQREF orKEREG at positions 43-47. Alternatively, also sequences such as TERE(for example TEREL), KECE (for example KECEL or KECER), RERE (forexample REREG), QERE (for example QEREG), KGRE (for example KGREG), KDRE(for example KDREV) are possible. Some other possible, but lesspreferred sequences include for example DECKL and NVCEL. ⁽⁴⁾With bothGLEW at positions 44-47 and KERE or KQRE at positions 43-46. ⁽⁵⁾Often asKP or EP at positions 83-84 of naturally occurring V_(HH) domains. ⁽⁶⁾Inparticular, but not exclusively, in combination with GLEW at positions44-47. ⁽⁷⁾With the proviso that when positions 44-47 are GLEW, position108 is always Q in (non-humanized) V_(HH) sequences that also contain aW at 103. ⁽⁸⁾The GLEW group also contains GLEW-like sequences atpositions 44-47, such as for example GVEW, EPEW, GLER, DQEW, DLEW, GIEW,ELEW, GPEW, EWLP, GPER, GLER and ELEW.

TABLE A-4 Some preferred but non-limiting combinations of HallmarkResidues in naturally occurring Nanobodies. 11 37 44 45 47 83 84 103 104108 DP-47 (human) M V G L W R A W G L “KERE” group L F E R L K P W G Q LF E R F E P W G Q L F E R F K P W G Q L Y Q R L K P W G Q L F L R V K PQ G Q L F Q R L K P W G Q L F E R F K P W G Q “GLEW” group L V G L W K SW G Q M V G L W K P R G Q For humanization of these combinations,reference is made to the specification.

In the Nanobodies, each amino acid residue at any other position thanthe Hallmark Residues can be any amino acid residue that naturallyoccurs at the corresponding position (according to the Kabat numbering)of a naturally occurring V_(HH) domain.

Such amino acid residues will be clear to the skilled person. Tables A-5to A-8 mention some non-limiting residues that can be present at eachposition (according to the Kabat numbering) of the FR1, FR2, FR3 and FR4of naturally occurring V_(HH) domains. For each position, the amino acidresidue that most frequently occurs at each position of a naturallyoccurring V_(HH) domain (and which is the most preferred amino acidresidue for said position in a Nanobody) is indicated in bold; and otherpreferred amino acid residues for each position have been underlined(note: the number of amino acid residues that are found at positions26-30 of naturally occurring V_(HH) domains supports the hypothesisunderlying the numbering by Chothia (supra) that the residues at thesepositions already form part of CDR1.)

In Tables A-5-A-8, some of the non-limiting residues that can be presentat each position of a human V_(H)3 domain have also been mentioned.Again, for each position, the amino acid residue that most frequentlyoccurs at each position of a naturally occurring human V_(H)3 domain isindicated in bold; and other preferred amino acid residues have beenunderlined.

For reference only, Tables A-5-A-8 also contain data on the V_(HH)entropy (“V_(HH) Ent”) and V_(HH) variability (“V_(HH) Var.”) at eachamino acid position for a representative sample of 1118 V_(HH) sequences(data kindly provided by David Lutje Hulsing and Prof. Theo Verrips ofUtrecht University). The values for the V_(HH) entropy and the V_(HH)variability provide a measure for the variability and degree ofconservation of amino acid residues between the 1118 V_(HH) sequencesanalyzed: low values (i.e. <1, such as <0.5) indicate that an amino acidresidue is highly conserved between the V_(HH) sequences (i.e. littlevariability). For example, the G at position 8 and the G at position 9have values for the V_(HH) entropy of 0.1 and 0 respectively, indicatingthat these residues are highly conserved and have little variability(and in case of position 9 is G in all 1118 sequences analysed), whereasfor residues that form part of the CDR's generally values of 1.5 or moreare found (data not shown). Note that (1) the amino acid residues listedin the second column of Tables A-5-A-8 are based on a bigger sample thanthe 1118 V_(HH) sequences that were analysed for determining the V_(HH)entropy and V_(HH) variability referred to in the last two columns; and(2) the data represented below support the hypothesis that the aminoacid residues at positions 27-30 and maybe even also at positions 93 and94 already form part of the CDR's (although the invention is not limitedto any specific hypothesis or explanation, and as mentioned above,herein the numbering according to Kabat is used). For a generalexplanation of sequence entropy, sequence variability and themethodology for determining the same, see Oliveira et al, PROTEINS:Structure, Function and Genetics, 52: 544-552 (2003).

TABLE A-5 Non-limiting examples of amino acid residues in FR1Amino acid residue(s): V_(HH) V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'sEnt. Var. 1 E, Q Q, A, E — — 2 V V 0.2 1 3 Q Q, K 0.3 2 4 L L 0.1 1 5V L Q, E, L, V 0.8 3 6 E E, D, Q, A 0.8 4 7 S, T S, F 0.3 2 8 G, R G 0.11 9 G G 0 1 10 G, V G, D, R 0.3 2 11 Hallmark residue: L, M, S, V, W; preferably L  0.8 2 12 V, I V, A 0.2 2 13 Q, K, RQ, E, K, P, R 0.4 4 14 P A, Q, A, G, P, S, T, V 1 5 15 G G 0 1 16 G, RG, A, E, D 0.4 3 17 S S, F 0.5 2 18 L L, V 0.1 1 19 R, KR, K, L, N, S, T 0.6 4 20 L L, F, I, V 0.5 4 21 S S, A, F, T 0.2 3 22 CC 0 1 23 A, T A, D, E, P, S, T, V 1.3 5 24 A A, I, L, S, T, V 1 6 25 SS, A, F, P, T 0.5 5 26 G G, A, D, E, R, S, T, V 0.7 7 27 FS, F, R, L, P, G, N, 2.3 13 28 T N, T, E, D, S, I, R, A, G, R, F, Y 1.711 29 F, V F, L, D, S, I, G, V, A 1.9 11 30 S, D, GN, S, E, G, A, D, M, T 1.8 11 (for the footnotes, see the footnotes toTable A-3)

TABLE A-6 Non-limiting examples of amino acid residues in FR2(for the footnotes, see the footnotes to TABLE A-3)Amino acid residue(s): V_(HH) V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'sEnt. Var. 36 W W 0.1 1 37Hallmark residue: F⁽¹⁾, H, I, L, Y or V, preferably F⁽¹⁾ or Y 1.1 6 38 RR 0.2 1 39 Q Q, H, P, R 0.3 2 40 A A, F, G, L, P, T, V 0.9 7 41 P, S, TP, A, L, S 0.4 3 42 G G, E 0.2 2 43 K K, D, E, N, Q, R, T, V 0.7 6 44Hallmark residue: G⁽²⁾, E⁽³⁾, A, D, Q, R, S, L; preferably G⁽²⁾, E⁽³⁾ 1.3 5 or Q; most preferably G⁽²⁾ or E⁽³⁾ 45Hallmark residue: L⁽²⁾, R 3) , C, I, L, P, Q, V; preferably L⁽²⁾ or R⁽³⁾0.6 4 46 E, V E, D, K, Q, V 0.4 2 47Hallmark residue: W⁽²⁾, L⁽¹⁾ or F⁽¹⁾, A, G, I, M, R, S, V or Y; 1.9 9preferably W⁽²⁾, L⁽¹⁾, F⁽¹⁾ or R 48 V V, I, L 0.4 3 49 S, A, GA, S, G, T, V 0.8 3

TABLE A-7 Non-limiting examples of amino acid residues in FR3(for the footnotes, see the footnotes to TABLE A-3)Amino acid residue(s): V_(HH) V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'sEnt. Var. 66 R R 0.1 1 67 F F, L, V 0.1 1 68 T T, A, N, S 0.5 4 69 II, L, M, V 0.4 4 70 S S, A, F, T 0.3 4 71 R R, G, H, I, L, K, Q, S, T, W1.2 8 72 D, E D, E, G, N, V 0.5 4 73 N, D, GN, A, D, F, I, K, L, R, S, T, V, Y 1.2 9 74 A, S A, D, G, N, P, S, T, V1 7 75 K K, A, E, K, L, N, Q, R 0.9 6 76 N, S N, D, K, R, S, T, Y 0.9 677 S, T, I T, A, E, I, M, P, S 0.8 5 78 L, A V, L, A, F, G, I, M 1.2 579 Y, H Y, A, D, F, H, N, S, T 1 7 80 L L, F, V 0.1 1 81 QQ, E, I, L, R, T 0.6 5 82 M M, I, L, V 0.2 2 82a N, G N, D, G, H, S, T0.8 4 82b S S, N, D, G, R, T 1 6 82c L L, P, V 0.1 2 83Hallmark residue: R, K⁽⁵⁾, N, E⁽⁵⁾, G, I, M, Q or T; preferably  0.9 7K or R; most preferably K 84Hallmark residue: P⁽⁵⁾, A, D, L, R, S, T, V; preferably P 0.7 6 85 E, GE, D, G, Q 0.5 3 86 D D 0 1 87 T, M T, A, S 0.2 3 88 A A, G, S 0.3 2 89V, L V, A, D, I, L, M, N, R, T 1.4 6 90 Y Y, F 0 1 91 Y, HY, D, F, H, L, S, T, V 0.6 4 92 C C 0 1 93 A, K, TA, N, G, H, K, N, R, S, T, V, Y 1.4 10 94 K, R, TA, V, C, F, G, I, K, L, R, S or T 1.6 9

TABLE A-8 Non-limiting examples of amino acid residues in FR4(for the footnotes, see the footnotes to TABLE A-3)Amino acid residue(s): V_(HH) V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'sEnt. Var. 103 Hallmark residue: W⁽⁴⁾, P⁽⁶⁾, R⁽⁶⁾, S; preferably W 0.4 2104 Hallmark residue: G or D; preferably G 0.1 1 105 Q, R Q, E, K, P, R0.6 4 106 G G 0.1 1 107 T T, A, I 0.3 2 108Hallmark residue: Q, L⁽⁷⁾, or R; preferably Q or L⁽⁷⁾ 0.4 3 109 V V 0.11 110 T T, I, A 0.2 1 111 V V, A, I 0.3 2 112 S S, F 0.3 1 113 SS, A, P, L, T 0.4 3

Thus, in another preferred, but not limiting aspect, a Nanobody of theinvention can be defined as an amino acid sequence with the (general)structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   i) one or more of the amino acid residues at positions 11, 37, 44,    45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering    are chosen from the Hallmark residues mentioned in Table A-3;    and in which:-   ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may behumanized Nanobodies. When the above Nanobody sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein.

In particular, a Nanobody of the invention can be an amino acid sequencewith the (general) structure

FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   i) (preferably) one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    A-3 (it being understood that V_(HH) sequences will contain one or    more Hallmark residues; and that partially humanized Nanobodies will    usually, and preferably, [still] contain one or more Hallmark    residues [although it is also within the scope of the invention to    provide—where suitable in accordance with the invention—partially    humanized Nanobodies in which all Hallmark residues, but not one or    more of the other amino acid residues, have been humanized]; and    that in fully humanized Nanobodies, where suitable in accordance    with the invention, all amino acid residues at the positions of the    Hallmark residues will be amino acid residues that occur in a human    V_(H)3 sequence. As will be clear to the skilled person based on the    disclosure herein that such V_(HH) sequences, such partially    humanized Nanobodies with at least one Hallmark residue, such    partially humanized Nanobodies without Hallmark residues and such    fully humanized Nanobodies all form aspects of this invention);    and in which:-   ii) said amino acid sequence has at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 1 to    22, in which for the purposes of determining the degree of amino    acid identity, the amino acid residues that form the CDR sequences    (indicated with X in the sequences of SEQ ID NO's: 1 to 22) are    disregarded;    and in which:-   iii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably    as defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may behumanized Nanobodies. When the above Nanobody sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein.

TABLE A-9Representative amino acid sequences for Nanobodies of the KERE, GLEW and P, R, S 103 group.The CDR's are indicated with XXXX KERE sequence no. 1 SEQ ID NO: 1EVQLVESGGGLVQPGGSLRLSCAASGIPFSXXXXXWFRQAPGKQRDSVAXXXXXRFTISRDNAKNTVYLQMNSLKPEDTAVYRCYFXXXXXWGQGTQVTVSS KERE sequence no. 2SEQ ID NO: 2 QVKLEESGGGLVQAGGSLRLSCVGSGRTFSXXXXXWFRLAPGKEREFVAXXXXXRFTISRDTASNRGYLHMNNLTPEDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 3SEQ ID NO: 3 AVQLVDSGGGLVQAGDSLKLSCALTGGAFTXXXXXWFRQTPGREREFVAXXXXXRFTISRDNAKNMVYLRMNSLIPEDAAVYSCAAXXXXXWGQGTLVTVSS KERE sequence no. 4SEQ ID NO: 4 QVQLVESGGGLVEAGGSLRLSCTASESPFRXXXXXWFRQTSGQEREFVAXXXXXRFTISRDDAKNTVWLHGSTLKPEDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 5SEQ ID NO: 5 AVQLVESGGGLVQGGGSLRLACAASERIFDXXXXXWYRQGPGNERELVAXXXXXRFTISMDYTKQTVYLHMNSLRPEDTGLYYCKIXXXXXWGQGTQVTVSS KERE sequence no. 6SEQ ID NO: 6 DVKFVESGGGLVQAGGSLRLSCVASGFNFDXXXXXWFRQAPGKEREEVAXXXXXRFTISSEKDKNSVYLQMNSLKPEDTALYICAGXXXXXWGRGTQVTVSS KERE sequence no. 7SEQ ID NO: 7 QVRLAESGGGLVQSGGSLRLSCVASGSTYTXXXXXWYRQYPGKQRALVAXXXXXRFTIARDSTKDTFCLQMNNLKPEDTAVYYCYAXXXXXWGQGTQVTVSS KERE sequence no. 8SEQ ID NO: 8 EVQLVESGGGLVQAGGSLRLSCAASGFTSDXXXXXWFRQAPGKPREGVSXXXXXRFTISTDNAKNTVHLLMNRVNAEDTALYYCAVXXXXXWGRGTRVTVSS KERE sequence no. 9SEQ ID NO: 9 QVQLVESGGGLVQPGGSLRLSCQASGDISTXXXXXWYRQVPGKLREFVAXXXXXRFTISGDNAKRAIYLQMNNLKPDOTAVYYCNRXXXXXWGQGTQVTVSP KERE sequence no. 10SEQ ID NO: 10 QVPVVESGGGLVQAGDSLRLFCAVPSFTSTXXXXXWFRQAPGKEREFVAXXXXXRFTISRNATKNTLTLRMDSLKPEDTAVYYCAAXXXXXWGDGTQVTVSS KERE sequence no. 11SEQ ID NO: 11 EVQLVESGGGLVQAGDSLRLFCTVSGGTASXXXXXWFRQAPGEKREFVAXXXXXRFTIARENAGNMVYLQMNNLKPDDTALYTCAAXXXXXWGRGTQVTVSS KERE sequence no. 12SEQ ID NO: 12 AVQLVESGGDSVQPGDSQTLSCAASGRTNSXXXXXWFRQAPGKERVFLAXXXXXRFTISRDSAKNMMYLQMNNLKPQDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 13SEQ ID NO: 13 AVQLVESGGGLVQAGGSLRLSCVVSGLTSSXXXXXWFRQTPWQERDFVAXXXXXRFTISRDNYKDTVLLEMNFLKPEDTAIYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 14SEQ ID NO: 14 AVQLVESGGGLVQAGASLRLSCATSTRTLDXXXXXWFRQAPGRDREFVAXXXXXRFTVSRDSAENTVALQMNSLKPEDTAVYYCAAXXXXXWGQGTRVTVSS KERE sequence no. 15SEQ ID NO: 15 QVQLVESGGGLVQPGGSLRLSCTVSRLTAHXXXXXWFRQAPGKEREAVSXXXXXRFTISRDYAGNTAFLQMDSLKPEDTGVYYCATXXXXXWGQGTQVTVSS KERE sequence no. 16SEQ ID NO: 16 EVQLVESGGELVQAGGSLKLSCTASGRNFVXXXXXWFRRAPGKEREFVAXXXXXRFTVSRDNGKNTAYLRMNSLKPEDTADYYCAVXXXXXLGSGTQVTVSS GLEW sequence no. 1SEQ ID NO: 17 AVQLVESGGGLVQPGGSLRLSCAASGFTFSXXXXXWVRQAPGKVLEWVSXXXXXRFTISRDNAKNTLYLQMNSLKPEDTAVYYCVKXXXXXGSQGTQVTVSS GLEW sequence no. 2SEQ ID NO: 18 EVQLVESGGGLVQPGGSLRLSCVCVSSGCTXXXXXWVRQAPGKAEEWVSXXXXXRFKISRDNAKKTLYLQMNSLGPEDTAMYYCQRXXXXXRGQGTQVTVSS GLEW sequence no. 3SEQ ID NO: 19 EVQLVESGGGLALPGGSLTLSCVFSGSTFSXXXXXWVRHTPGKAEEWVSXXXXXRFTISRDNAKNTLYLEMNSLSPEDTAMYYCGRXXXXXRSKGIQVTVSS P, R, S 103 sequence no. 1SEQ ID NO: 20 AVQLVESGGGLVQAGGSLRLSCAASGRTFSXXXXXWFRQAPGKEREFVAXXXXXRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAXXXXXRGQGTQVTVSS P, R, S 103 sequence no. 2SEQ ID NO: 21 DVQLVESGGDLVQPGGSLRLSCAASGFSFDXXXXXWLRQTPGKGLEWVGXXXXXRFTISRDNAKNMLYLHLNNLKSEDTAVYYCRRXXXXXLGQGTQVTVSS P, R, S 103 sequence no. 3SEQ ID NO: 22 EVQLVESGGGLVQPGGSLRLSCVCVSSGCTXXXXXWVRQAPGKAEEWVSXXXXXRFKISRDNAKKTLYLQMNSLGPEDTAMYYCQRXXXXXRGQGTQVTVSS

In particular, a Nanobody of the invention of the KERE group can be anamino acid sequence with the (general) structure

FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4in which:

-   i) the amino acid residue at position 45 according to the Kabat    numbering is a charged amino acid (as defined herein) or a cysteine    residue, and position 44 is preferably an E;    and in which:-   ii) FR1 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-10Representative FW1 sequences for Nanobodies of the KERE-group.KERE FW1 sequence no. 1 SEQ ID NO: 23 QVQRVESGGGLVQAGGSLRLSCAASGRTSSKERE FW1 sequence no. 2 SEQ ID NO: 24 QVQLVESGGGLVQTGDSLSLSCSASGRTFSKERE FW1 sequence no. 3 SEQ ID NO: 25 QVKLEESGGGLVQAGDSLRLSCAATGRAFGKERE FW1 sequence no. 4 SEQ ID NO: 26 AVQLVESGGGLVQPGESLGLSCVASGRDFVKERE FW1 sequence no. 5 SEQ ID NO: 27 EVQLVESGGGLVQAGGSLRLSCEVLGRTAGKERE FW1 sequence no. 6 SEQ ID NO: 28 QVQLVESGGGWVQPGGSLRLSCAASETILSKERE FW1 sequence no. 7 SEQ ID NO: 29 QVQLVESGGGTVQPGGSLNLSCVASGNTFNKERE FW1 sequence no. 8 SEQ ID NO: 30 EVQLVESGGGLAQPGGSLQLSCSAPGFTLDKERE FW1 sequence no. 9 SEQ ID NO: 31 AQELEESGGGLVQAGGSLRLSCAASGRTFNand in which:

-   iii) FR2 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-11 Representative FW2 sequences for Nanobodies of the KERE-group. KERE FW2  SEQ ID NO: 41 WFRQAPGKEREFVA sequence no. 1KERE FW2  SEQ ID NO: 42 WFRQTPGREREFVA sequence no. 2 KERE FW2 SEQ ID NO: 43 WYRQAPGKQREMVA sequence no. 3 KERE FW2  SEQ ID NO: 44WYRQGPGKQRELVA sequence no. 4 KERE FW2  SEQ ID NO: 45 WIRQAPGKEREGVSsequence no. 5 KERE FW2  SEQ ID NO: 46 WFREAPGKEREGIS sequence no. 6KERE FW2  SEQ ID NO: 47 WYRQAPGKERDLVA sequence no. 7 KERE FW2 SEQ ID NO: 48 WFRQAPGKQREEVS sequence no. 8 KERE FW2  SEQ ID NO: 49WFRQPPGKVREFVG sequence no. 9and in which:

-   iv) FR3 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-12Representative FW3 sequences for Nanobodies of the KERE-group.KERE FW3 sequence no. 1 SEQ ID NO: 50 RFTISRDNAKNTVYLQMNSLKPEDTAVYRCYFKERE FW3 sequence no. 2 SEQ ID NO: 51 RFAISRDNNKNTGYLQMNSLEPEDTAVYYCAAKERE FW3 sequence no. 3 SEQ ID NO: 52 RFTVARNNAKNTVNLEMNSLKPEDTAVYYCAAKERE FW3 sequence no. 4 SEQ ID NO: 53 RFTISRDIAKNTVDLLMNNLEPEDTAVYYCAAKERE FW3 sequence no. 5 SEQ ID NO: 54 RLTISRDNAVDTMYLQMNSLKPEDTAVYYCAAKERE FW3 sequence no. 6 SEQ ID NO: 55 RFTISRDNAKNTVYLQMDNVKPEDTAIYYCAAKERE FW3 sequence no. 7 SEQ ID NO: 56 RFTISKDSGKNTVYLQMTSLKPEDTAVYYCATKERE FW3 sequence no. 8 SEQ ID NO: 57 RFTISRDSAKNMMYLQMNNLKPQDTAVYYCAAKERE FW3 sequence no. 9 SEQ ID NO: 58 RFTISRENDKSTVYLQLNSLKPEDTAVYYCAAKERE FW3 sequence no. 10 SEQ ID NO: 59 RFTISRDYAGNTAYLQMNSLKPEDTGVYYCATand in which:

-   v) FR4 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-13 Representative FW4 sequences forNanobodies of the KERE-group. KERE FW4  SEQ ID NO: 60 WGQGTQVTVSSsequence no. 1 KERE FW4  SEQ ID NO: 61 WGKGTLVTVSS sequence no. 2KERE FW4  SEQ ID NO: 62 RGQGTRVTVSS sequence no. 3 KERE FW4 SEQ ID NO: 63 WGLGTQVTISS sequence no. 4and in which:

-   vi) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In the above Nanobodies, one or more of the further Hallmark residuesare preferably as described herein (for example, when they are V_(HH)sequences or partially humanized Nanobodies).

Also, the above Nanobodies may for example be V_(HH) sequences or may behumanized Nanobodies. When the above Nanobody sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein.

With regard to framework 1, it will be clear to the skilled person that,when an amino acid sequence as outlined above is generated by expressionof a nucleotide sequence, the first four amino acid sequences (i.e.amino acid residues 1-4 according to the Kabat numbering) may often bedetermined by the primer(s) that have been used to generate said nucleicacid. Thus, for determining the degree of amino acid identity, the firstfour amino acid residues are preferably disregarded.

Also, with regard to framework 1, and although amino acid positions 27to 30 are according to the Kabat numbering considered to be part of theframework regions (and not the CDR's), it has been found by analysis ofa database of more than 1000 V_(HH) sequences that the positions 27 to30 have a variability (expressed in terms of V_(HH) entropy and V_(HH)variability—see Tables A-5 to A-8) that is much greater than thevariability on positions 1 to 26. Because of this, for determining thedegree of amino acid identity, the amino acid residues at positions 27to 30 are preferably also disregarded.

In view of this, a Nanobody of the KERE class may be an amino acidsequence that is comprised of four framework regions/sequencesinterrupted by three complementarity determining regions/sequences, inwhich:

-   i) the amino acid residue at position 45 according to the Kabat    numbering is a charged amino acid (as defined herein) or a cysteine    residue, and position 44 is preferably an E;    and in which:-   ii) FR1 is an amino acid sequence that, on positions 5 to 26 of the    Kabat numbering, has at least 80% amino acid identity with at least    one of the following amino acid sequences:

TABLE A-14 Representative FW1 sequences (amino acid residues5 to 26) for Nanobodies of the KERE-group. KERE FW1 sequence no. 10SEQ ID NO: 32 VESGGGLVQPGGSLRLSCAASG KERE FW1 sequence no. 11SEQ ID NO: 33 VDSGGGLVQAGDSLKLSCALTG KERE FW1 sequence no. 12SEQ ID NO: 34 VDSGGGLVQAGDSLRLSCAASG KERE FW1 sequence no. 13SEQ ID NO: 35 VDSGGGLVEAGGSLRLSCQVSE KERE FW1 sequence no. 14SEQ ID NO: 36 QDSGGGSVQAGGSLKLSCAASG KERE FW1 sequence no. 15SEQ ID NO: 37 VQSGGRLVQAGDSLRLSCAASE KERE FW1 sequence no. 16SEQ ID NO. 38 VESGGTLVQSGDSLKLSCASST KERE FW1 sequence no. 17SEQ ID NO: 39 MESGGDSVQSGGSLTLSCVASG KERE FW1 sequence no. 18SEQ ID NO: 40 QASGGGLVQAGGSLRLSCSASVand in which:

-   iii) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4    of Nanobodies of the KERE-class:    and in which:-   iv) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may behumanized Nanobodies. When the above Nanobody sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein.

A Nanobody of the GLEW class may be an amino acid sequence that iscomprised of four framework regions/sequences interrupted by threecomplementarity determining regions/sequences, in which

-   i) preferably, when the Nanobody of the GLEW-class is a    non-humanized Nanobody, the amino acid residue in position 108 is Q;-   ii) FR1 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-15Representative FW1 sequences for Nanobodies of the GLEW-group.GLEW FW1 sequence no. 1 SEQ ID NO: 64 QVQLVESGGGLVQPGGSLRLSCAASGFTFSGLEW FW1 sequence no. 2 SEQ ID NO: 65 EVHLVESGGGLVRPGGSLRLSCAAFGFIFKGLEW FW1 sequence no. 3 SEQ ID NO: 66 QVKLEESGGGLAQPGGSLRLSCVASGFTFSGLEW FW1 sequence no. 4 SEQ ID NO: 67 EVOLVESGGGLVQPGGSLRLSCVCVSSGCTGLEW FW1 sequence no. 5 SEQ ID NO: 68 EVQLVESGGGLALPGGSLTLSCVFSGSTFSand in which:

-   iii) FR2 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-16 Representative FW2 sequences for Nanobodiesof the GLEW-group. GLEW FW2  SEQ ID NO: 72 WVRQAPGKAEEWVS sequence no. 1GLEW FW2  SEQ ID NO: 73 WVRRPPGKGLEWVS sequence no. 2 GLEW FW2 SEQ ID NO: 74 WVRQAPGMGLEWVS sequence no. 3 GLEW FW2  SEQ ID NO: 75WVRQAPGKEPEWVS sequence no. 4 GLEW FW2  SEQ ID NO: 76 WVRQAPGKDQEWVSsequence no. 5 GLEW FW2  SEQ ID NO: 77 WVRQAPGKAEEWVS sequence no. 6GLEW FW2  SEQ ID NO: 78 WVRQAPGKGLEWVA sequence no. 7 GLEW FW2 SEQ ID NO: 79 WVRQAPGRATEWVS sequence no. 8and in which:

-   iv) FR3 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-17Representative FW3 sequences for Nanobodies of the GLEW-group.GLEW FW3 sequence no. 1 SEQ ID NO: 80 RFTISRDNAKNTLYLQMNSLKPEDTAVYYCVKGLEW FW3 sequence no. 2 SEQ ID NO: 81 RFTISRDNARNTLYLQMDSLIPEDTALYYCARGLEW FW3 sequence no. 3 SEQ ID NO: 82 RFTSSRDNAKSTLYLQMNDLKPEDTALYYCARGLEW FW3 sequence no. 4 SEQ ID NO: 83 RFIISRDNAKNTLYLQMNSLGPEDTAMYYCQRGLEW FW3 sequence no. 5 SEQ ID ND: 84 RFTASRDNAKNTLYLQMNSLKSEDTARYYCARGLEW FW3 sequence no. 6 SEQ ID NO: 85 RFTISRDNAKNTLYLQMDDLQSEDTAMYYCGRand in which:

-   v) FR4 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-18 Representative FW4 sequences forNanobodies of the GLEW-group. GLEW FW4  SEQ ID NO: 86 GSQGTQVTVSSsequence no. 1 GLEW FW4  SEQ ID NO: 87 LRGGTQVTVSS sequence no. 2GLEW FW4  SEQ ID NO: 88 RGQGTLVTVSS sequence no. 3 GLEW FW4 SEQ ID NO: 89 RSRGIQVTVSS sequence no. 4 GLEW FW4  SEQ ID NO: 90WGKGTQVTVSS sequence no. 5 GLEW FW4  SEQ ID NO: 91 WGQGTQVTVSSsequence no. 6and in which:

-   vi) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In the above Nanobodies, one or more of the further Hallmark residuesare preferably as described herein (for example, when they are V_(HH)sequences or partially humanized Nanobodies).

With regard to framework 1, it will again be clear to the skilled personthat, for determining the degree of amino acid identity, the amino acidresidues on positions 1 to 4 and 27 to 30 are preferably disregarded.

In view of this, a Nanobody of the GLEW class may be an amino acidsequence that is comprised of four framework regions/sequencesinterrupted by three complementarity determining regions/sequences, inwhich:

-   i) preferably, when the Nanobody of the GLEW-class is a    non-humanized Nanobody, the amino acid residue in position. 108 is    Q;    and in which:-   ii) FR1 is an amino acid sequence that, on positions 5 to 26 of the    Kabat numbering, has at least 80% amino acid identity with at least    one of the following amino acid sequences:

TABLE A-19 Representative FW1 sequences (amino acid residues5 to 26) for Nanobodies of the KERE-group. GLEW FW1 sequence no. 6SEQ ID NO: 69 VESGGGLVQPGGSLRLSCAASG GLEW FW1 sequence no. 7SEQ ID NO: 70 EESGGGLAQPGGSLRLSCVASG GLEW FW1 sequence no. 8SEQ ID NO: 71 VESGGGLALPGGSLTLSCVFSGand in which:

-   iii) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4    of Nanobodies of the GLEW-class;    and in which:-   iv) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may behumanized Nanobodies. When the above Nanobody sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein. Inthe above Nanobodies, one or more of the further Hallmark residues arepreferably as described herein (for example, when they are V_(HH)sequences or partially humanized Nanobodies).

A Nanobody of the P, R, S 103 class may be an amino acid sequence thatis comprised of four framework regions/sequences interrupted by threecomplementarity determining regions/sequences, in which

-   i) the amino acid residue at position 103 according to the Kabat    numbering is different from W;    and in which:-   ii) preferably the amino acid residue at position 103 according to    the Kabat numbering is P, R or S, and more preferably R;    and in which:-   iii) FR1 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-20Representative FW1 sequences for Nanobodies of the P, R, S 103-group.P, R, S 103 FW1 sequence no. 1 SEQ ID NO: 92AVQLVESGGGLVQAGGSLRLSCAASGRTFS P, R, S 103 FW1 sequence no. 2SEQ ID NO: 93 QVQLQESGGGMVQPGGSLRLSCAASGFDFGP, R, S 103 FW1 sequence no. 3 SEQ ID NO: 94EVHLVESGGGLVRPGGSLRLSCAAFGFIFK P, R, S 103 FW1 sequence no. 4SEQ ID NO: 95 QVQLAESGGGLVQPGGSLKLSCAASRTIVSP, R, S 103 FW1 sequence no. 5 SEQ ID NO: 96QEHLVESGGGLVDIGGSLRLSCAASERIFS P, R, S 103 FW1 sequence no. 6SEQ ID NO: 97 QVKLEESGGGLAQPGGSLRLSCVASGFTFSP, R, S 103 FW1 sequence no. 7 SEQ ID NO: 98EVQLVESGGGLVQPGGSLRLSCVCVSSGCT P, R, S 103 FW1 sequence no. 8SEQ ID NO: 99 EVQLVESGGGLALPGGSLTLSCVFSGSTFSand in which

-   iv) FR2 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-21 Representative FW2 sequences for Nanobodiesof the P, R, S 103-group. P, R, S 103 FW2 sequence no. 1 SEQ ID NO: 102WFRQAPGKEREFVA P, R, S 103 FW2 sequence no. 2 SEQ ID NO: 103WVRQAPGKVLEWVS P, R, S 103 FW2 sequence no. 3 SEQ ID NO: 104WVRRPPGKGLEWVS P, R, S 103 FW2 sequence no. 4 SEQ ID NO: 105WIRQAPGKEREGVS P, R, S 103 FW2 sequence no. 5 SEQ ID NO: 106WVRQYPGKEPEWVS P, R, S 103 FW2 sequence no. 6 SEQ ID NO: 107WFRQPPGKEHEFVA P, R, S 103 FW2 sequence no. 7 SEQ ID NO: 108WYRQAPGKRTELVA P, R, S 103 FW2 sequence no. 8 SEQ ID NO: 109WLRQAPGQGLEWVS P, R, S 103 FW2 sequence no. 9 SEQ ID NO: 110WLRQTPGKGLEWVG P, R, S 103 FW2 sequence no. 10 SEQ ID NO: 111WVRQAPGKAEEFVSand in which:

-   v) FR3 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-22 Representative FW3 sequences for Nanobodies of the P, R, S103-group. P, R, S 103 FW3 SEQ ID NO: 112RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA sequence no. 1 P, R, S 103 FW3SEQ ID NO: 113 RFTISRDNARNTLYLQMDSLIPEDTALYYCAR sequence no. 2P, R, S 103 FW3 SEQ ID NO: 114 RFTISRDNAKNEMYLQMNNLKTEDTGVYWCGAsequence no. 3 P, R, S 103 FW3 SEQ ID NO: 115RFTISSDSNRNMIYLQMNNLKPEDTAVYYCAA sequence no. 4 P, R, S 103 FW3SEQ ID NO: 116 RFTISRDNAKNMLYLHLNNLKSEDTAVYYCRR sequence no. 5P, R, S 103 FW3 SEQ ID NO: 117 RFTISRDNAKKTVYLRLNSLNPEDTAVYSCNLsequence no. 6 P, R, S 103 FW3 SEQ ID NO: 118RFKISRDNAKKTLYLQMNSLGPEDTAMYYCQR sequence no. 7 P, R, S 103 FW3SEQ ID NO: 119 RFTVSRDNGKNTAYLRMNSLKPEDTADYYCAV sequence no. 8and in which:

-   vi) FR4 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-23 Representative FW4 sequences for Nanobodiesof the P, R, S 103-group. P, R, S 103 FW4 SEQ ID NO: 120 RGQGTQVTVSSsequence no. 1 P, R, S 103 FW4 SEQ ID NO: 121 LRGGTQVTVSS sequence no. 2P, R, S 103 FW4 SEQ ID NO: 122 GNKGTLVTVSS sequence no. 3P, R, S 103 FW4 SEQ ID NO: 123 SSPGTQVTVSS sequence no. 4P, R, S 103 FW4 SEQ ID NO: 124 SSQGTLVTVSS sequence no. 5P, R, S 103 FW4 SEQ ID NO: 125 RSRGIQVTVSS sequence no. 6and in which:

-   vii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably    as defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In the above Nanobodies, one or more of the further Hallmark residuesare preferably as described herein (for example, when they are V_(HH)sequences or partially humanized Nanobodies).

With regard to framework 1, it will again be clear to the skilled personthat, for determining the degree of amino acid identity, the amino acidresidues on positions 1 to 4 and 27 to 30 are preferably disregarded.

In view of this, a Nanobody of the P,R,S 103 class may be an amino acidsequence that is comprised of four framework regions/sequencesinterrupted by three complementarity determining regions/sequences, inwhich:

-   i) the amino acid residue at position 103 according to the Kabat    numbering is different from W;    and in which:-   ii) preferably the amino acid residue at position 103 according to    the Kabat numbering is P, R or S, and more preferably R;    and in which:-   iii) FR1 is an amino acid sequence that, on positions 5 to 26 of the    Kabat numbering, has at least 80% amino acid identity with at least    one of the following amino acid sequences:

TABLE A-24 Representative FW1 sequences (amino acidresidues 5 to 26) for Nanobodies of the P, R, S 103-group.P, R, S 103 FW1 SEQ ID NO: 100 VESGGGLVQAGGSLRL sequence no. 9 SCAASGP, R, S 103 FW1 SEQ ID NO: 101 AESGGGLVQPGGSLKL sequence no. 10 SCAASRand in which:

-   iv) FR2. FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4 of    Nanobodies of the P,R,S 103 class;    and in which:-   v) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may behumanized Nanobodies. When the above Nanobody sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein.

In the above Nanobodies, one or more of the further Hallmark residuesare preferably as described herein (for example, when they are V_(HH)sequences or partially humanized Nanobodies).

In another preferred, but non-limiting aspect, the invention relates toa Nanobody as described above, in which the CDR sequences have at least70% amino acid identity, preferably at least 80% amino acid identity,more preferably at least 90% amino acid identity, such as 95% amino acididentity or more or even essentially 100% amino acid identity with theCDR sequences of at least one of the amino acid sequences of SEQ IDNO's: 455 to 501, more preferably 455 to 457, 459, 460, 464 to 469. Thisdegree of amino acid identity can for example be determined bydetermining the degree of amino acid identity (in a manner describedherein) between said Nanobody and one or more of the sequences of SEQ IDNO's: 455 to 501, more preferably 455 to 457, 459, 460, 464 to 469, inwhich the amino acid residues that form the framework regions aredisregarded. Such Nanobodies can be as further described herein.

As already mentioned herein, another preferred but non-limiting aspectof the invention relates to a Nanobody with an amino acid sequence thatis chosen from the group consisting of SEQ ID NO's: 455 to 501, morepreferably 455 to 457, 459, 460, 464 to 469 or from the group consistingof from amino acid sequences that have more than 80%, preferably morethan 90%, more preferably more than 95%, such as 99% or more sequenceidentity (as defined herein) with at least one of the amino acidsequences of SEQ ID NO's: 455 to 501, more preferably 455 to 457, 459,460, 464 to 469.

Also, in the above Nanobodies:

-   i) any amino acid substitution (when it is not a humanizing    substitution as defined herein) is preferably, and compared to the    corresponding amino acid sequence of SEQ ID NO's: 455 to 501, more    preferably 455 to 457, 459, 460, 464 to 469, a conservative amino    acid substitution, (as defined herein);    and/or:-   ii) its amino acid sequence preferably contains either only amino    acid substitutions, or otherwise preferably no more than 5,    preferably no more than 3, and more preferably only 1 or 2 amino    acid deletions or insertions, compared to the corresponding amino    acid sequence of SEQ ID NO's: 455 to 501, more preferably 455 to    457, 459, 460, 464 to 469;    and/or-   iii) the CDR's may be CDR's that are derived by means of affinity    maturation, for example starting from the CDR's of to the    corresponding amino acid sequence of SEQ ID NO's: 455 to 501, more    preferably 455 to 457, 459, 460, 464 to 469.

Preferably, the CDR sequences and FR sequences in the Nanobodies of theinvention are such that the Nanobodies of the invention (andpolypeptides of the invention comprising the same):

-   -   bind to Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,        Angptl3, Angptl4, Angptl5, or Angptl6, more preferably Tie2,        Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2 or Ang2 with        a dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or        less, and preferably 10⁻⁷ to 10⁻¹² moles/liter or less and more        preferably 10⁻⁸ to 10⁻¹² moles/liter (i.e. with an association        constant (K_(A)) of 10⁵ to 10¹² liter/moles or more, and        preferably 10⁷ to 10¹² liter/moles or more and more preferably        10⁸ to 10¹² liter/moles);        and/or such that they:    -   bind to Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,        Angptl3, Angptl4, Angptl5, or Angptl6, more preferably Tie2,        Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2 or Ang2 with        a k_(on)-rate of between 10² M⁻¹s⁻¹ to about 10⁷M⁻¹s⁻¹,        preferably between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more preferably        between 10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹        and 10⁷ M⁻¹s⁻¹;        and/or such that they:    -   bind to Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,        Angptl3, Angptl4, Angptl5, or Angptl6, more preferably Tie2,        Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2 or Ang2 with        a k_(off) rate between 1s⁻¹ (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹        (providing a near irreversible complex with a t_(1/2) of        multiple days), preferably between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more        preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as between 10⁻⁴        s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, CDR sequences and FR sequences present in the Nanobodies ofthe invention are such that the Nanobodies of the invention will bind toTie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4,Angptl5, or Angptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4,more preferably Tie2 or Ang2 with an affinity less than 500 nM,preferably less than 200 nM, more preferably less than 10 nM, such asless than 500 pM.

According to one non-limiting aspect of the invention, a Nanobody may beas defined herein, but with the proviso that it has at least “one aminoacid difference” (as defined herein) in at least one of the frameworkregions compared to the corresponding framework region of a naturallyoccurring human V_(H) domain, and in particular compared to thecorresponding framework region of DP-47. More specifically, according toone non-limiting aspect of the invention, a Nanobody may be as definedherein, but with the proviso that it has at least “one amino aciddifference” (as defined herein) at least one of the Hallmark residues(including those at positions 108, 103 and/or 45) compared to thecorresponding framework region of a naturally occurring human V_(H)domain, and in particular compared to the corresponding framework regionof DP-47. Usually, a Nanobody will have at least one such amino aciddifference with a naturally occurring V_(H) domain in at least one ofFR2 and/or FR4, and in particular at least one of the Hallmark residuesin FR2 and/or FR4 (again, including those at positions 108, 103 and/or45).

Also, a humanized Nanobody of the invention may be as defined herein,but with the proviso that it has at least “one amino acid difference”(as defined herein) in at least one of the framework regions compared tothe corresponding framework region of a naturally occurring V_(HH)domain. More specifically, according to one non-limiting aspect of theinvention, a humanized Nanobody may be as defined herein, but with theproviso that it has at least “one amino acid difference” (as definedherein) at least one of the Hallmark residues (including those atpositions 108, 103 and/or 45) compared to the corresponding frameworkregion of a naturally occurring V_(HH) domain. Usually, a humanizedNanobody will have at least one such amino acid difference with anaturally occurring V_(HH) domain in at least one of FR2 and/or FR4, andin particular at least one of the Hallmark residues in FR2 and/or FR4(again, including those at positions 108, 103 and/or 45).

As will be clear from the disclosure herein, it is also within the scopeof the invention to use natural or synthetic analogs, mutants, variants,alleles, homologs and orthologs (herein collectively referred to as“analogs”) of the Nanobodies of the invention as defined herein, and inparticular analogs of the Nanobodies of SEQ ID NO's 455 to 501, morepreferably 455 to 457, 459, 460, 464 to 469. Thus, according to oneaspect of the invention, the term “Nanobody of the invention” in itsbroadest sense also covers such analogs.

Generally, in such analogs, one or more amino acid residues may havebeen replaced, deleted and/or added, compared to the Nanobodies of theinvention as defined herein. Such substitutions, insertions or deletionsmay be made in one or more of the framework regions and/or in one ormore of the CDR's. When such substitutions, insertions or deletions aremade in one or more of the framework regions, they may be made at one ormore of the Hallmark residues and/or at one or more of the otherpositions in the framework residues, although substitutions, insertionsor deletions at the Hallmark residues are generally less preferred(unless these are suitable humanizing substitutions as describedherein).

By means of non-limiting examples, a substitution may for example be aconservative substitution (as described herein) and/or an amino acidresidue may be replaced by another amino acid residue that naturallyoccurs at the same position in another V_(HH) domain (see Tables A-5 toA-8 for some non-limiting examples of such substitutions), although theinvention is generally not limited thereto. Thus, any one or moresubstitutions, deletions or insertions, or any combination thereof, thateither improve the properties of the Nanobody of the invention or thatat least do not detract too much from the desired properties or from thebalance or combination of desired properties of the Nanobody of theinvention (i.e. to the extent that the Nanobody is no longer suited forits intended use) are included within the scope of the invention. Askilled person will generally be able to determine and select suitablesubstitutions, deletions or insertions, or suitable combinations ofthereof, based on the disclosure herein and optionally after a limiteddegree of routine experimentation, which may for example involveintroducing a limited number of possible substitutions and determiningtheir influence on the properties of the Nanobodies thus obtained.

For example, and depending on the host organism used to express theNanobody or polypeptide of the invention, such deletions and/orsubstitutions may be designed in such a way that one or more sites forpost-translational modification (such as one or more glycosylationsites) are removed, as will be within the ability of the person skilledin the art. Alternatively, substitutions or insertions may be designedso as to introduce one or more sites for attachment of functional groups(as described herein), for example to allow site-specific pegylation(again as described herein).

As can be seen from the data on the V_(HH) entropy and V_(HH)variability given in Tables A-5 to A-8 above, some amino acid residuesin the framework regions are more conserved than others. Generally,although, the invention in its broadest sense is not limited thereto,any substitutions, deletions or insertions are preferably made atpositions that are less conserved. Also, generally, amino acidsubstitutions are preferred over amino acid deletions or insertions.

The analogs are preferably such that they can bind to Tie1, Tie2, Ang1,Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, morepreferably Tie2 or Ang2 with an affinity (suitably measured and/orexpressed as a K_(D)-value (actual or apparent), a K_(A)-value (actualor apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively asan IC₅₀ value, as further described herein) that is as defined hereinfor the Nanobodies of the invention.

The analogs are preferably also such that they retain the favourableproperties the Nanobodies, as described herein.

Also, according to one preferred aspect, the analogs have a degree ofsequence identity of at least 70%, preferably at least 80%, morepreferably at least 90%, such as at least 95% or 99% or more; and/orpreferably have at most 20, preferably at most 10, even more preferablyat most 5, such as 4, 3, 2 or only 1 amino acid difference (as definedherein), with one of the Nanobodies of SEQ ID NOs: 455 to 501, morepreferably 455 to 457, 459, 460, 464 to 469.

Also, the framework sequences and CDR's of the analogs are preferablysuch that they are in accordance with the preferred aspects definedherein. More generally, as described herein, the analogs will have (a) aQ at position 108; and/or (b) a charged amino acid or a cysteine residueat position 45 and preferably an E at position 44, and more preferably Eat position 44 and R at position 45; and/or (c) P, R or S at position103.

One preferred class of analogs of the Nanobodies of the inventioncomprise Nanobodies that have been humanized (i.e. compared to thesequence of a naturally occurring Nanobody of the invention). Asmentioned in the background art cited herein, such humanizationgenerally involves replacing one or more amino acid residues in thesequence of a naturally occurring V_(HH) with the amino acid residuesthat occur at the same position in a human V_(H) domain, such as a humanV_(H)3 domain. Examples of possible humanizing substitutions orcombinations of humanizing substitutions will be clear to the skilledperson, for example from the Tables herein, from the possible humanizingsubstitutions mentioned in the background art cited herein, and/or froma comparison between the sequence of a Nanobody and the sequence of anaturally occurring human V_(H) domain.

The humanizing substitutions should be chosen such that the resultinghumanized Nanobodies still retain the favourable properties ofNanobodies as defined herein, and more preferably such that they are asdescribed for analogs in the preceding paragraphs. A skilled person willgenerally be able to determine and select suitable humanizingsubstitutions or suitable combinations of humanizing substitutions,based on the disclosure herein and optionally after a limited degree ofroutine experimentation, which may for example involve introducing alimited number of possible humanizing substitutions and determiningtheir influence on the properties of the Nanobodies thus obtained.

Generally, as a result of humanization, the Nanobodies of the inventionmay become more “human-like”, while still retaining the favourableproperties of the Nanobodies of the invention as described herein. As aresult, such humanized Nanobodies may have several advantages, such as areduced immunogenicity, compared to the corresponding naturallyoccurring V_(HH) domains. Again, based on the disclosure herein andoptionally after a limited degree of routine experimentation, theskilled person will be able to select humanizing substitutions orsuitable combinations of humanizing substitutions which optimize orachieve a desired or suitable balance between the favourable propertiesprovided by the humanizing substitutions on the one hand and thefavourable properties of naturally occurring V_(HH) domains on the otherhand.

The Nanobodies of the invention may be suitably humanized at anyframework residue(s), such as at one or more Hallmark residues (asdefined herein) or at one or more other framework residues (i.e.non-Hallmark residues) or any suitable combination thereof. Onepreferred humanizing substitution for Nanobodies of the “P,R,S-103group” or the “KERE group” is Q108 into L108. Nanobodies of the “GLEWclass” may also be humanized by a Q108 into L108 substitution, providedat least one of the other Hallmark residues contains a Camelid(camelizing) substitution (as defined herein). For example, as mentionedabove, one particularly preferred class of humanized Nanobodies has GLEWor a GLEW-like sequence at positions 44-47; P, R or S (and in particularR) at position 103, and an L at position 108.

The humanized and other analogs, and nucleic acid sequences encoding thesame, can be provided in any manner known per se. For example, theanalogs can be obtained by providing a nucleic acid that encodes anaturally occurring V_(HH) domain, changing the codons for the one ormore amino acid residues that are to be substituted into the codons forthe corresponding desired amino acid residues (e.g. by site-directedmutagenesis or by PCR using suitable mismatch primers), expressing thenucleic acid/nucleotide sequence thus obtained in a suitable host orexpression system; and optionally isolating and/or purifying the analogthus obtained to provide said analog in essentially isolated form (e.g.as further described herein). This can generally be performed usingmethods and techniques known per se, which will be clear to the skilledperson, for example from the handbooks and references cited herein, thebackground art cited herein and/or from the further description herein.Alternatively, a nucleic acid encoding the desired analog can besynthesized in a manner known per se (for example using an automatedapparatus for synthesizing nucleic acid sequences with a predefinedamino acid sequence) and can then be expressed as described herein. Yetanother technique may involve combining one or more naturally occurringand/or synthetic nucleic acid sequences each encoding a part of thedesired analog, and then expressing the combined nucleic acid sequenceas described herein. Also, the analogs can be provided using chemicalsynthesis of the pertinent amino acid sequence using techniques forpeptide synthesis known per se, such as those mentioned herein.

In this respect, it will be also be clear to the skilled person that theNanobodies of the invention (including their analogs) can be designedand/or prepared starting from human V_(H) sequences (i.e. amino acidsequences or the corresponding nucleotide sequences), such as forexample from human V_(H)3 sequences such as DP-47, DP-51 or DP-29, i.e.by introducing one or more camelizing substitutions (i.e. changing oneor more amino acid residues in the amino acid sequence of said humanV_(H) domain into the amino acid residues that occur at thecorresponding position in a V_(HH) domain), so as to provide thesequence of a Nanobody of the invention and/or so as to confer thefavourable properties of a Nanobody to the sequence thus obtained.Again, this can generally be performed using the various methods andtechniques referred to in the previous paragraph, using an amino acidsequence and/or nucleotide sequence for a human V_(H) domain as astarting point.

Some preferred, but non-limiting camelizing substitutions can be derivedfrom Tables A-5-A-8. It will also be clear that camelizing substitutionsat one or more of the Hallmark residues will generally have a greaterinfluence on the desired properties than substitutions at one or more ofthe other amino acid positions, although both and any suitablecombination thereof are included within the scope of the invention. Forexample, it is possible to introduce one or more camelizingsubstitutions that already confer at least some the desired properties,and then to introduce further camelizing substitutions that eitherfurther improve said properties and/or confer additional favourableproperties. Again, the skilled person will generally be able todetermine and select suitable camelizing substitutions or suitablecombinations of camelizing substitutions, based on the disclosure hereinand optionally after a limited degree of routine experimentation, whichmay for example involve introducing a limited number of possiblecamelizing substitutions and determining whether the favourableproperties of Nanobodies are obtained or improved (i.e. compared to theoriginal Y domain). Generally, however, such camelizing substitutionsare preferably such that the resulting an amino acid sequence at leastcontains (a) a Q at position 108; and/or (b) a charged amino acid or acysteine residue at position 45 and preferably also an E at position 44,and more preferably E at position 44 and R at position 45; and/or (c) P,R or S at position 103; and optionally one or more further camelizingsubstitutions. More preferably, the camelizing substitutions are suchthat they result in a Nanobody of the invention and/or in an analogthereof (as defined herein), such as in a humanized analog and/orpreferably in an analog that is as defined in the preceding paragraphs.

As will also be clear from the disclosure herein, it is also within thescope of the invention to use parts or fragments, or combinations of twoor more parts or fragments, of the Nanobodies of the invention asdefined herein, and in particular parts or fragments of the Nanobodiesof SEQ ID NQ's: 455 to 501, more preferably 455 to 457, 459, 460, 464 to469. Thus, according to one aspect of the invention, the term “Nanobodyof the invention” in its broadest sense also covers such parts orfragments.

Generally, such parts or fragments of the Nanobodies of the invention(including analogs thereof) have amino acid sequences in which, comparedto the amino acid sequence of the corresponding full length Nanobody ofthe invention (or analog thereof), one or more of the amino acidresidues at the N-terminal end, one or more amino acid residues at theC-terminal end, one or more contiguous internal amino acid residues, orany combination thereof, have been deleted and/or removed.

The parts or fragments are preferably such that they can bind to Tie1,Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4,Angptl5, or Angptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4,more preferably Tie2 or Ang2 with an affinity (suitably measured and/orexpressed as a K_(D)-value (actual or apparent), a K_(A)-value (actualor apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively asan IC₅₀ value, as further described herein) that is as defined hereinfor the Nanobodies of the invention.

Any part or fragment is preferably such that it comprises at least 10contiguous amino acid residues, preferably at least 20 contiguous aminoacid residues, more preferably at least 30 contiguous amino acidresidues, such as at least 40 contiguous amino acid residues, of theamino acid sequence of the corresponding full length Nanobody of theinvention.

Also, any part or fragment is such preferably that it comprises at leastone of CDR1, CDR2 and/or CDR3 or at least part thereof (and inparticular at least CDR3 or at least part thereof). More preferably, anypart or fragment is such that it comprises at least one of the CDR's(and preferably at least CDR3 or part thereof) and at least one otherCDR (i.e. CDR1 or CDR2) or at least part thereof, preferably connectedby suitable framework sequence(s) or at least part thereof. Morepreferably, any part or fragment is such that it comprises at least oneof the CDR's (and preferably at least CDR3 or part thereof) and at leastpart of the two remaining CDR's, again preferably connected by suitableframework sequence(s) or at least part thereof.

According to another particularly preferred, but non-limiting aspect,such a part or fragment comprises at least CDR3, such as FR3, CDR3 andFR4 of the corresponding full length Nanobody of the invention, i.e. asfor example described in the International application WO 03/050531(tasters et al.).

As already mentioned above, it is also possible to combine two or moreof such parts or fragments (i.e. from the same or different Nanobodiesof the invention), i.e. to provide an analog (as defined herein) and/orto provide further parts or fragments (as defined herein) of a Nanobodyof the invention. It is for example also possible to combine one or moreparts or fragments of a Nanobody of the invention with one or more partsor fragments of a human V_(H) domain.

According to one preferred aspect, the parts or fragments have a degreeof sequence identity of at least 50%, preferably at least 60%, morepreferably at least 70%, even more preferably at least 80%, such as atleast 90%, 95% or 99% or more with one of the Nanobodies of SEQ ID NOs455 to 501, more preferably 455 to 457, 459, 460, 464 to 469.

The parts and fragments, and nucleic acid sequences encoding the same,can be provided and optionally combined in any manner known per se. Forexample, such parts or fragments can be obtained by inserting a stopcodon in a nucleic acid that encodes a full-sized Nanobody of theinvention, and then expressing the nucleic acid thus obtained in amanner known per se (e.g. as described herein). Alternatively, nucleicacids encoding such parts or fragments can be obtained by suitablyrestricting a nucleic acid that encodes a full-sized Nanobody of theinvention or by synthesizing such a nucleic acid in a manner known perse. Parts or fragments may also be provided using techniques for peptidesynthesis known per se.

The invention in its broadest sense also comprises derivatives of theNanobodies of the invention. Such derivatives can generally be obtainedby modification, and in particular by chemical and/or biological (e.g.enzymatic) modification, of the Nanobodies of the invention and/or ofone or more of the amino acid residues that form the Nanobodies of theinvention.

Examples of such modifications, as well as examples of amino acidresidues within the Nanobody sequence that can be modified in such amanner (i.e. either on the protein backbone but preferably on a sidechain), methods and techniques that can be used to introduce suchmodifications and the potential uses and advantages of suchmodifications will be clear to the skilled person.

For example, such a modification may involve the introduction (e.g. bycovalent linking or in an other suitable manner) of one or morefunctional groups, residues or moieties into or onto the Nanobody of theinvention, and in particular of one or more functional groups, residuesor moieties that confer one or more desired properties orfunctionalities to the Nanobody of the invention. Example of suchfunctional groups will be clear to the skilled person.

For example, such modification may comprise the introduction (e.g. bycovalent binding or in any other suitable manner) of one or morefunctional groups that increase the half-life, the solubility and/or theabsorption of the Nanobody of the invention, that reduce theimmunogenicity and/or the toxicity of the Nanobody of the invention,that eliminate or attenuate any undesirable side effects of the Nanobodyof the invention, and/or that confer other advantageous properties toand/or reduce the undesired properties of the Nanobodies and/orpolypeptides of the invention; or any combination of two or more of theforegoing. Examples of such functional groups and of techniques forintroducing them will be clear to the skilled person, and can generallycomprise all functional groups and techniques mentioned in the generalbackground art cited hereinabove as well as the functional groups andtechniques known per se for the modification of pharmaceutical proteins,and in particular for the modification of antibodies or antibodyfragments (including ScFv's and single domain antibodies), for whichreference is for example made to Remington's Pharmaceutical Sciences,16th ed., Mack Publishing Co., Easton, Pa. (1980). Such functionalgroups may for example be linked directly (for example covalently) to aNanobody of the invention, or optionally via a suitable linker orspacer, as will again be clear to the skilled person.

One of the most widely used techniques for increasing the half-lifeand/or reducing the immunogenicity of pharmaceutical proteins comprisesattachment of a suitable pharmacologically acceptable polymer, such aspoly(ethyleneglycol) (PEG) or derivatives thereof (such asmethoxypoly(ethyleneglycol) or mPEG). Generally, any suitable form ofpegylation can be used, such as the pegylation used in the art forantibodies and antibody fragments (including but not limited to (single)domain antibodies and ScFv's); reference is made to for example Chapman,Nat. Biotechnol., 54, 531-545 (2002); by Veronese and Harris, Adv. DrugDeliv. Rev. 54, 453-456 (2003), by Harris and Chess, Nat. Rev. Drug.Discov., 2, (2003) and in WO 04/060965. Various reagents for pegylationof proteins are also commercially available, for example from NektarTherapeutics, USA.

Preferably, site-directed pegylation is used, in particular via acysteine-residue (see for example Yang et al., Protein Engineering, 16,10, 761-770 (2003). For example, for this purpose, PEG may be attachedto a cysteine residue that naturally occurs in a Nanobody of theinvention, a Nanobody of the invention may be modified so as to suitablyintroduce one or more cysteine residues for attachment of PEG, or anamino acid sequence comprising one or more cysteine residues forattachment of PEG may be fused to the N- and/or C-terminus of a Nanobodyof the invention, all using techniques of protein engineering known perse to the skilled person.

Preferably, for the Nanobodies and proteins of the invention, a PEG isused with a molecular weight of more than 5000, such as more than 10,000and less than 200,000, such as less than 100,000; for example in therange of 20,000-80,000.

Another, usually less preferred modification comprises N-linked orO-linked glycosylation, usually as part of co-translational and/orpost-translational modification, depending on the host cell used forexpressing the Nanobody or polypeptide of the invention.

Yet another modification may comprise the introduction of one or moredetectable labels or other signal-generating groups or moieties,depending on the intended use of the labelled Nanobody. Suitable labelsand techniques for attaching, using and detecting them will be clear tothe skilled person, and for example include, but are not limited to,fluorescent labels (such as fluorescein, isothiocyanate, rhodamine,phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, andfluorescamine and fluorescent metals such as ¹⁵²Eu or others metals fromthe lanthanide series), phosphorescent labels, chemiluminescent labelsor bioluminescent labels (such as luminal, isoluminol, theromaticacridinium ester, imidazole, acridinium salts, oxalate ester, dioxetaneor GFP and its analogs), radio-isotopes (such as ³H, ¹²⁵I, ³²P, ³⁵S,¹⁴C, ⁵¹Cr, ³⁶Cl, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, and ⁷⁵Se), metals, metal chelates ormetallic cations (for example metallic cations such as ^(99m)Tc ¹²³I,¹¹¹In, ¹³¹I, ⁹⁷Ru, ⁶⁷Cu, ⁶⁷Ga, and ⁶⁸Ga or other metals or metalliccations that are particularly suited for use in in vivo, in vitro or insitu diagnosis and imaging, such as (¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr, and⁵⁶Fe), as well as chromophores and enzymes (such as malatedehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeastalcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, biotinavidin peroxidase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase,glucoamylase and acetylcholine esterase). Other suitable labels will beclear to the skilled person, and for example include moieties that canbe detected using NMR or ESR spectroscopy.

Such labelled Nanobodies and polypeptides of the invention may forexample be used for in vitro, in vivo or in situ assays (includingimmunoassays known per se such as ELISA, RIA, EIA and other “sandwichassays”, etc.) as well as in vivo diagnostic and imaging purposes,depending on the choice of the specific label.

As will be clear to the skilled person, another modification may involvethe introduction of a chelating group, for example to chelate one of themetals or metallic cations referred to above. Suitable chelating groupsfor example include, without limitation, diethyl-enetriaminepentaaceticacid (DTPA) or ethylenediaminetetraacetic acid. (EDTA).

Yet another modification may comprise the introduction of a functionalgroup that is one part of a specific binding pair, such as thebiotin-(strept)avidin binding pair. Such a functional group may be usedto link the Nanobody of the invention to another protein, polypeptide orchemical compound that is bound to the other half of the binding pair,i.e. through formation of the binding pair. For example, a Nanobody ofthe invention may be conjugated to biotin, and linked to anotherprotein, polypeptide, compound or carrier conjugated to avidin orstreptavidin. For example, such a conjugated Nanobody may be used as areporter, for example in a diagnostic system where a detectablesignal-producing agent is conjugated to avidin or streptavidin. Suchbinding pairs may for example also be used to bind the Nanobody of theinvention to a carrier, including carriers suitable for pharmaceuticalpurposes. One non-limiting example are the liposomal formulationsdescribed by Cao and Suresh, Journal of Drug Targetting, 8, 4, 257(2000). Such binding pairs may also be used to link a therapeuticallyactive agent to the Nanobody of the invention.

For some applications, in particular for those applications in which itis intended to kill a cell that expresses the target against which theNanobodies of the invention are directed (e.g. in the treatment ofcancer), or to reduce or slow the growth and/or proliferation such acell, the Nanobodies of the invention may also be linked to a toxin orto a toxic residue or moiety. Examples of toxic moieties, compounds orresidues which can be linked to a Nanobody of the invention toprovide—for example—a cytotoxic compound will be clear to the skilledperson and can for example be found in the prior art cited above and/orin the further description herein. One example is the so-called ADEPT™technology described in WO 03/055527.

Other potential chemical and enzymatical modifications will be clear tothe skilled person. Such modifications may also be introduced forresearch purposes (e.g. to study function-activity relationships).Reference is for example made to Lundblad and Bradshaw, Biotechnol.Appl. Biochem., 26, 143-151 (1997).

Preferably, the derivatives are such that they bind to Tie1, Tie2, Ang1,Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, morepreferably Tie2 or Ang2 with an affinity (suitably measured and/orexpressed as a K_(D)-value (actual or apparent), a K_(A)-value (actualor apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively asan IC₅₀ value, as further described herein) that is as defined hereinfor the Nanobodies of the invention.

As mentioned above, the invention also relates to proteins orpolypeptides that essentially consist of or comprise at least oneNanobody of the invention. By “essentially consist of” is meant that theamino acid sequence of the polypeptide of the invention either isexactly the same as the amino acid sequence of a Nanobody of theinvention or corresponds to the amino acid sequence of a Nanobody of theinvention which has a limited number of amino acid residues, such as1-20 amino acid residues, for example 1-10 amino acid residues andpreferably 1-6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 aminoacid residues, added at the amino terminal end, at the carboxy terminalend, or at both the amino terminal end and the carboxy terminal end ofthe amino acid sequence of the Nanobody.

Said amino acid residues may or may not change, alter or otherwiseinfluence the (biological) properties of the Nanobody and may or may notadd further functionality to the Nanobody. For example, such amino acidresidues:

-   -   can comprise an N-terminal Met residue, for example as result of        expression in a heterologous host cell or host organism.    -   may form a signal sequence or leader sequence that directs        secretion of the Nanobody from a host cell upon synthesis.        Suitable secretory leader peptides will be clear to the skilled        person, and may be as further described herein. Usually, such a        leader sequence will be linked to the N-terminus of the        Nanobody, although the invention in its broadest sense is not        limited thereto;    -   may form a sequence or signal that allows the Nanobody to be        directed towards and/or to penetrate or enter into specific        organs, tissues, cells, or parts or compartments of cells,        and/or that allows the Nanobody to penetrate or cross a        biological barrier such as a cell membrane, a cell layer such as        a layer of epithelial cells, a tumour including solid tumours,        or the blood-brain-barrier. Examples of such amino acid        sequences will be clear to the skilled person. Some non-limiting        examples are the small peptide vectors (“Pep-trans vectors”)        described in WO 03/026700 and in Temsamani et al., Expert Opin.        Biol. Ther., 1, 773 (2001); Temsamani and Vidal, Drug Discov.        Today, 9, 1012 (004) and Rousselle, J. Pharmacol. Exp. Ther.,        296, 124-131 (2001), and the membrane translocator sequence        described by Zhao et al., Apoptosis, 8, 631-637 (2003).        C-terminal and N-terminal amino acid sequences for intracellular        targeting of antibody fragments are for example described by        Cardinale et al., Methods, 34, 171 (2004). Other suitable        techniques for intracellular targeting involve the expression        and/or use of so-called “intrabodies” comprising a Nanobody of        the invention, as mentioned below;    -   may form a “tag”, for example an amino acid sequence or residue        that allows or facilitates the purification of the Nanobody, for        example using affinity techniques directed against said sequence        or residue. Thereafter, said sequence or residue may be removed        (e.g. by chemical or enzymatical cleavage) to provide the        Nanobody sequence (for this purpose, the tag may optionally be        linked to the Nanobody sequence via a cleavable linker sequence        or contain a cleavable motif). Some preferred, but non-limiting        examples of such residues are multiple histidine residues,        glutatione residues and a myc-tag (see for example SEQ ID NO:31        of WO 06/12282).    -   may be one or more amino acid residues that have been        functionalized and/or that can serve as a site for attachment of        functional groups. Suitable amino acid residues and functional        groups will be clear to the skilled person and include, but are        not limited to, the amino acid residues and functional groups        mentioned herein for the derivatives of the Nanobodies of the        invention.

According to another aspect, a polypeptide of the invention comprises aNanobody of the invention, which is fused at its amino terminal end, atits carboxy terminal end, or both at its amino terminal end and at itscarboxy terminal end to at least one further amino acid sequence, i.e.so as to provide a fusion protein comprising said Nanobody of theinvention and the one or more further amino acid sequences. Such afusion will also be referred to herein as a “Nanobody fusion”.

The one or more further amino acid sequence may be any suitable and/ordesired amino acid sequences. The further amino acid sequences may ormay not change, alter or otherwise influence the (biological) propertiesof the Nanobody, and may or may not add further functionality to theNanobody or the polypeptide of the invention. Preferably, the furtheramino acid sequence is such that it confers one or more desiredproperties or functionalities to the Nanobody or the polypeptide of theinvention.

For example, the further amino acid sequence may also provide a secondbinding site, which binding site may be directed against any desiredprotein, polypeptide, antigen, antigenic determinant or epitope(including but not limited to the same protein, polypeptide, antigen,antigenic determinant or epitope against which the Nanobody of theinvention is directed, or a different protein, polypeptide, antigen,antigenic determinant or epitope).

Example of such amino acid sequences will be clear to the skilledperson, and may generally comprise all amino acid sequences that areused in peptide fusions based on conventional antibodies and fragmentsthereof (including but not limited to ScFv's and single domainantibodies). Reference is for example made to the review by Holliger andHudson, Nature Biotechnology, 23, 9, 1126-1136 (2005).

For example, such an amino acid sequence may be an amino acid sequencethat increases the half-life, the solubility, or the absorption, reducesthe immunogenicity or the toxicity, eliminates or attenuates undesirableside effects, and/or confers other advantageous properties to and/orreduces the undesired properties of the polypeptides of the invention,compared to the Nanobody of the invention per se. Some non-limitingexamples of such amino acid sequences are serum proteins, such as humanserum albumin (see for example WO 00/27435) or haptenic molecules (forexample haptens that are recognized by circulating antibodies, see forexample WO 98/22141).

In particular, it has been described in the art that linking fragmentsof immunoglobulins (such as V_(H) domains) to serum albumin or tofragments thereof can be used to increase the half-life. Reference isfor made to WO 00/27435 and WO 01/077137). According to the invention,the Nanobody of the invention is preferably either directly linked toserum albumin (or to a suitable fragment thereof) or via a suitablelinker, and in particular via a suitable peptide linked so that thepolypeptide of the invention can be expressed as a genetic fusion(protein). According to one specific aspect, the Nanobody of theinvention may be linked to a fragment of serum albumin that at leastcomprises the domain III of serum albumin or part thereof. Reference isfor example made to the U.S. provisional application 60/788,256 ofAblynx N.V. entitled “Albumin derived amino acid sequence, use thereoffor increasing the half-life of therapeutic proteins and of othertherapeutic proteins and entities, and constructs comprising the same”filed on Mar. 31, 2006.

Alternatively, the further amino acid sequence may provide a secondbinding site or binding unit that is directed against a serum protein(such as, for example, human serum albumin or another serum protein suchas IgG), so as to provide increased half-life in serum. Such amino acidsequences for example include the Nanobodies described below, as well asthe small peptides and binding proteins described in WO 91/01743, WO01/45746 and WO 02/076489 and the dAb's described in WO 03/002609 and WO04/003019. Reference is also made to Harmsen et al., Vaccine, 23 (41);4926-42, 2005, as well as to EP 0 368 684, as well as to the followingthe U.S. provisional applications 60/843,349, 60/850,774, 60/850,775 byAblynx N.V. mentioned herein and US provisional application of AblynxN.V. entitled “Peptides capable of binding to serum proteins” filed onDec. 5, 2006 (also mentioned herein).

Such amino acid sequences may in particular be directed against serumalbumin (and more in particular human serum albumin) and/or against IgG(and more in particular human IgG). For example, such amino acidsequences may be amino acid sequences that are directed against (human)serum albumin and amino acid sequences that can bind to amino acidresidues on (human) serum albumin that are not involved in binding ofserum albumin to FcRn (see for example WO 06/0122787) and/or amino acidsequences that are capable of binding to amino acid residues on serumalbumin that do not form part of domain III of serum albumin (see againfor example WO 06/0122787); amino acid sequences that have or canprovide an increased half-life (see for example the U.S. provisionalapplication 60/843,349 by Ablynx N.V. entitled “Serum albumin bindingproteins with long half-lives” filed on Sep. 8, 2006); amino acidsequences against human serum albumin that are cross-reactive with serumalbumin from at least one species of mammal, and in particular with atleast one species of primate (such as, without limitation, monkeys fromthe genus Macaca (such as, and in particular, cynomologus monkeys(Macaca fascicularis) and/or rhesus monkeys (Macaca mulatta)) and baboon(Papio ursinus), reference is again made to the U.S. provisionalapplication 60/843,349); amino acid sequences that can bind to serumalbumin in a pH independent manner (see for example the U.S. provisionalapplication 60/850,774 by Ablynx N.V. entitled “Amino acid sequencesthat bind to serum proteins in a manner that is essentially independentof the pH, compounds comprising the same, and uses thereof”, filed onOct. 11, 2006) and/or amino acid sequences that are conditional binders(see for example the U.S. provisional application 60/850,775 by AblynxN.V. entitled “Amino acid sequences that bind to a desired molecule in aconditional manner”, filed on Oct. 11, 2006).

According to another aspect, the one or more further amino acidsequences may comprise one or more parts, fragments or domains ofconventional 4-chain antibodies (and in particular human antibodies)and/or of heavy chain antibodies. For example, although usually lesspreferred, a Nanobody of the invention may be linked to a conventional(preferably human) V_(H) or V_(L) domain or to a natural or syntheticanalog of a V_(H) or V_(L) domain, again optionally via a linkersequence (including but not limited to other (single) domain antibodies,such as the dAb's described by Ward et al.).

The at least one Nanobody may also be linked to one or more (preferablyhuman) C_(H)1 C_(H)2 and/or C_(H)3 domains, optionally via a linkersequence. For instance, a Nanobody linked to a suitable C_(H)1 domaincould for example be used—together with suitable light chains—togenerate antibody fragments/structures analogous to conventional Fabfragments or F(ab′)₂ fragments, but in which one or (in case of anF(ab′)₂ fragment) one or both of the conventional V_(H) domains havebeen replaced by a Nanobody of the invention. Also, two Nanobodies couldbe linked to a C_(H)3 domain (optionally via a linker) to provide aconstruct with increased half-life in vivo.

According to one specific aspect of a polypeptide of the invention, oneor more Nanobodies of the invention may be linked to one or moreantibody parts, fragments or domains that confer one or more effectorfunctions to the polypeptide of the invention and/or may confer theability to bind to one or more Fc receptors. For example, for thispurpose, and without being limited thereto, the one or more furtheramino acid sequences may comprise one or more C_(H)2 and/or C_(H)3domains of an antibody, such as from a heavy chain antibody (asdescribed herein) and more preferably from a conventional human 4-chainantibody; and/or may form (part of) and Fc region, for example from IgG,from IgE or from another human Ig. For example, WO 94/04678 describesheavy chain antibodies comprising a Camelid V_(HH) domain or a humanizedderivative thereof (i.e. a Nanobody), in which the Camelidae C_(H)2and/or C_(H)3 domain have been replaced by human C_(H)2 and C_(H)3domains, so as to provide an immunoglobulin that consists of 2 heavychains each comprising a Nanobody and human C_(H)2 and C_(H)3 domains(but no C_(H)1 domain), which immunoglobulin has the effector functionprovided by the C_(H)2 and C_(H)3 domains and which immunoglobulin canfunction without the presence of any light chains. Other amino acidsequences that can be suitably linked to the Nanobodies of the inventionso as to provide an effector function will be clear to the skilledperson, and may be chosen on the basis of the desired effectorfunction(s). Reference is for example made to WO 04/058820, WO 99/42077and WO 05/017148, as well as the review by Holliger and Hudson, supra.Coupling of a Nanobody of the invention to an Fc portion may also leadto an increased half-life, compared to the corresponding Nanobody of theinvention. For some applications, the use of an Fc portion and/or ofconstant domains (i.e. C_(H)2 and/or C_(H)3 domains) that conferincreased half-life without any biologically significant effectorfunction may also be suitable or even preferred. Other suitableconstructs comprising one or more Nanobodies and one or more constantdomains with increased half-life in vivo will be clear to the skilledperson, and may for example comprise two Nanobodies linked to a C_(H)3domain, optionally via a linker sequence. Generally, any fusion proteinor derivatives with increased half-life will preferably have a molecularweight of more than 50 kD, the cut-off value for renal absorption.

The further amino acid sequences may also folio a signal sequence orleader sequence that directs secretion of the Nanobody or thepolypeptide of the invention from a host cell upon synthesis (forexample to provide a pre-, pro- or prepro-form of the polypeptide of theinvention, depending on the host cell used to express the polypeptide ofthe invention).

The further amino acid sequence may also form a sequence or signal thatallows the Nanobody or polypeptide of the invention to be directedtowards and/or to penetrate or enter into specific organs, tissues,cells, or parts or compartments of cells, and/or that allows theNanobody or polypeptide of the invention to penetrate or cross abiological barrier such as a cell membrane, a cell layer such as a layerof epithelial cells, a tumor including solid tumours, or theblood-brain-barrier. Suitable examples of such amino acid sequences willbe clear to the skilled person, and for example include, but are notlimited to, the “Peptrans” vectors mentioned above, the sequencesdescribed by Cardinale et al. and the amino acid sequences and antibodyfragments known per se that can be used to express or produce theNanobodies and polypeptides of the invention as so-called “intrabodies”,for example as described in WO 94/02610, WO 95/22618, U.S. Pat. No.7,004,940, WO 03/014960, WO 99/07414; WO 05/01690; EP 1 512 696; and inCattaneo, A. & Biocca, S. (1997) Intracellular Antibodies: Developmentand Applications. Landes and Springer-Verlag; and in Kontermann, Methods34, (2004), 163-170, and the further references described therein.

For some applications, in particular for those applications in which itis intended to kill a cell that expresses the target against which theNanobodies of the invention are directed (e.g. in the treatment ofcancer), or to reduce or slow the growth and/or proliferation of such acell, the Nanobodies of the invention may also be linked to a(cyto)toxic protein or polypeptide. Examples of such toxic proteins andpolypeptides which can be linked to a Nanobody of the invention toprovide—for example—a cytotoxic polypeptide of the invention will beclear to the skilled person and can for example be found in the priorart cited above and/or in the further description herein. One example isthe so-called ADEPT™ technology described in WO 03/055527.

According to one preferred, but non-limiting aspect, said one or morefurther amino acid sequences comprise at least one further Nanobody, soas to provide a polypeptide of the invention that comprises at leasttwo, such as three, four, five or more Nanobodies, in which saidNanobodies may optionally be linked via one or more linker sequences (asdefined herein). Polypeptides of the invention that comprise two or moreNanobodies, of which at least one is a Nanobody of the invention, willalso be referred to herein as “multivalent” polypeptides of theinvention, and the Nanobodies present in such polypeptides will also bereferred to herein as being in a “multivalent format”. For example a“bivalent” polypeptide of the invention comprises two Nanobodies,optionally linked via a linker sequence, whereas a “trivalent”polypeptide of the invention comprises three Nanobodies, optionallylinked via two linker sequences; etc.; in which at least one of theNanobodies present in the polypeptide, and up to all of the Nanobodiespresent in the polypeptide, is/are a Nanobody of the invention.

In a multivalent polypeptide of the invention, the two or moreNanobodies may be the same or different, and may be directed against thesame antigen or antigenic determinant (for example against the samepart(s) or epitope(s) or against different parts or epitopes) or mayalternatively be directed against different antigens or antigenicdeterminants; or any suitable combination thereof. For example, abivalent polypeptide of the invention may comprise (a) two identicalNanobodies; (b) a first Nanobody directed against a first antigenicdeterminant of a protein or antigen and a second Nanobody directedagainst the same antigenic determinant of said protein or antigen whichis different from the first Nanobody; (c) a first Nanobody directedagainst a first antigenic determinant of a protein or antigen and asecond Nanobody directed against another antigenic determinant of saidprotein or antigen; or (d) a first Nanobody directed against a firstprotein or antigen and a second Nanobody directed against a secondprotein or antigen (i.e. different from said first antigen). Similarly,a trivalent polypeptide of the invention may, for example and withoutbeing limited thereto. comprise (a) three identical Nanobodies; (b) twoidentical Nanobody against a first antigenic determinant of an antigenand a third Nanobody directed against a different antigenic determinantof the same antigen; (c) two identical Nanobody against a firstantigenic determinant of an antigen and a third Nanobody directedagainst a second antigen different from said first antigen; (d) a firstNanobody directed against a first antigenic determinant of a firstantigen, a second Nanobody directed against a second antigenicdeterminant of said first antigen and a third Nanobody directed againsta second antigen different from said first antigen; or (e) a firstNanobody directed against a first antigen, a second Nanobody directedagainst a second antigen different from said first antigen, and a thirdNanobody directed against a third antigen different from said first andsecond antigen.

Polypeptides of the invention that contain at least two Nanobodies, inwhich at least one Nanobody is directed against a first antigen (i.e.against Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3,Angptl4, Angptl5, or Angptl6, more preferably Tie2, Ang2, Ang1, Ang4, orAngptl4, more preferably Tie2 or Ang2,) and at least one Nanobody isdirected against a second antigen (i.e. different from Tie1, Tie2, Ang1,Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, morepreferably Tie2 or Ang2,), will also be referred to as “multispecific”polypeptides of the invention, and the Nanobodies present in suchpolypeptides will also be referred to herein as being in a“multispecific format”. Thus, for example, a “bispecific” polypeptide ofthe invention is a polypeptide that comprises at least one Nanobodydirected against a first antigen (i.e. Tie1, Tie2, Ang1, Ang2, Ang3,Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, morepreferably Tie2, Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2 orAng2,) and at least one further Nanobody directed against a secondantigen (i.e. different from Tie1, Tie2, Ang1, Ang2, Ang3, Ang4,Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, more preferablyTie2, Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2 or Ang2,),whereas a “trispecific” polypeptide of the invention is a polypeptidethat comprises at least one Nanobody directed against a first antigen(i.e. Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3,Angptl4, Angptl5, or Angptl6, more preferably Tie2, Ang2, Ang1, Ang4, orAngptl4, more preferably Tie2 or Ang2,), at least one further Nanobodydirected against a second antigen (i.e. different from Tie1, Tie2, Ang1,Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, morepreferably Tie2 or Ang2,) and at least one further Nanobody directedagainst a third antigen (i.e. different from both. Tie1, Tie2, Ang1,Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, morepreferably Tie2 or Ang2, and the second antigen); etc.

Accordingly, in its simplest form, a bispecific polypeptide of theinvention is a bivalent polypeptide of the invention (as definedherein), comprising a first Nanobody directed against Tie1, Tie2, Ang1,Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, morepreferably Tie2 or Ang2, and a second Nanobody directed against a secondantigen, in which said first and second Nanobody may optionally belinked via a linker sequence (as defined herein); whereas a trispecificpolypeptide of the invention in its simplest form is a trivalentpolypeptide of the invention (as defined herein), comprising a firstNanobody directed against Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1,Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, more preferably Tie2,Ang2, Ang3, Ang4, or Angptl4, more preferably Tie2 or Ang2, a secondNanobody directed against a second antigen and a third Nanobody directedagainst a third antigen, in which said first, second and third Nanobodymay optionally be linked via one or more, and in particular one andmore, in particular two, linker sequences.

However, as will be clear from the description hereinabove, theinvention is not limited thereto, in the sense that a multispecificpolypeptide of the invention may comprise at least one Nanobody againstTie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4,Angptl5, or Angptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4,more preferably Tie2 or Ang2, and any number of Nanobodies directedagainst one or more antigens different from Tie1, Tie2, Ang1, Ang2,Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6,more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2or Ang2.

Furthermore, although it is encompassed within the scope of theinvention that the specific order or arrangement of the variousNanobodies in the polypeptides of the invention may have some influenceon the properties of the final polypeptide of the invention (includingbut not limited to the affinity, specificity or avidity for Tie1, Tie2,Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, morepreferably Tie2 or Ang2, or against the one or more other antigens),said order or arrangement is usually not critical and may be suitablychosen by the skilled person, optionally after some limited routineexperiments based on the disclosure herein. Thus, when reference is madeto a specific multivalent or multispecific polypeptide of the invention,it should be noted that this encompasses any order or arrangements ofthe relevant Nanobodies, unless explicitly indicated otherwise.

Finally, it is also within the scope of the invention that thepolypeptides of the invention contain two or more Nanobodies and one ormore further amino acid sequences (as mentioned herein).

For multivalent and multispecific polypeptides containing one or moreV_(HH) domains and their preparation, reference is also made to Conrathet al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001; Muyldermans,Reviews in Molecular Biotechnology 74 (2001), 277-302; as well as to forexample WO 96/34103 and WO 99/23221. Some other examples of somespecific multispecific and/or multivalent polypeptide of the inventioncan be found in the applications by Ablynx N.V. referred to herein.

One preferred, but non-limiting example of a multispecific polypeptideof the invention comprises at least one Nanobody of the invention and atleast one Nanobody that provides for an increased half-life. SuchNanobodies may for example be Nanobodies that are directed against aserum protein, and in particular a human serum protein, such as humanserum albumin, thyroxine-binding protein, (human) transferrin,fibrinogen, an immunoglobulin such as IgG, IgE or IgM, or against one ofthe serum proteins listed in WO 04/003019. Of these, Nanobodies that canbind to serum albumin (and in particular human serum albumin) or to IgG(and in particular human IgG, see for example Nanobody VH-1 described inthe review by Muyldermans, supra) are particularly preferred (althoughfor example, for experiments in mice or primates, Nanobodies against orcross-reactive with mouse serum albumin (MSA) or serum albumin from saidprimate, respectively, can be used. However, for pharmaceutical use,Nanobodies against human serum albumin or human IgG will usually bepreferred). Nanobodies that provide for increased half-life and that canbe used in the polypeptides of the invention include the Nanobodiesdirected against serum albumin that are described in WO 04/041865, in WO06/122787 and in the further patent applications by Ablynx N.V., such asthose mentioned above.

For example, the some preferred Nanobodies that provide for increasedhalf-life for use in the present invention include Nanobodies that canbind to amino acid residues on (human) serum albumin that are notinvolved in binding of serum albumin to FcRn (see for example WO06/0122787); Nanobodies that are capable of binding to amino acidresidues on serum albumin that do not form part of domain III of serumalbumin (see for example WO 06/0122787); Nanobodies that have or canprovide an increased half-life (see for example the U.S. provisionalapplication 60/843,349 by Ablynx N.V mentioned herein); Nanobodiesagainst human serum albumin that are cross-reactive with serum albuminfrom at least one species of mammal, and in particular with at least onespecies of primate (such as, without limitation, monkeys from the genusMacaca (such as, and in particular, cynomologus monkeys (Macacafascicularis) and/or rhesus monkeys (Macaca mulatta)) and baboon (Papioursinus)) (see for example the U.S. provisional application 60/843,349by Ablynx N.V); Nanobodies that can bind to serum albumin in a pHindependent manner (see for example the U.S. provisional application60/850,774 by Ablynx. N.V. mentioned herein) and/or Nanobodies that areconditional binders (see for example the U.S. provisional application60/850,775 by Ablynx N.V.).

Some particularly preferred Nanobodies that provide for increasedhalf-life and that can be used in the polypeptides of the inventioninclude the Nanobodies ALB-1 to ALB-10 disclosed in WO 06/122787 (seeTables II and III) of which ALB-8 (SEQ ID NO: 62 in WO 06/122787) isparticularly preferred.

Some preferred, but non-limiting examples of polypeptides of theinvention that comprise at least one Nanobody of the invention and atleast one Nanobody that provides for increased half-life.

According to a specific, but non-limiting aspect of the invention, thepolypeptides of the invention contain, besides the one or moreNanobodies of the invention, at least one Nanobody against human serumalbumin.

Generally, any polypeptides of the invention with increased half-lifethat contain one or more Nanobodies of the invention, and anyderivatives of Nanobodies of the invention or of such polypeptides thathave an increased half-life, preferably have a half-life that is atleast 1.5 times, preferably at least 2 times, such as at least 5 times,for example at least 10 times or more than 20 times, greater than thehalf-life of the corresponding Nanobody of the invention per se. Forexample, such a derivative or polypeptides with increased half-life mayhave a half-life that is increased with more than 1 hours, preferablymore than 2 hours, more preferably more than 6 hours, such as more than12 hours, or even more than 24, 48 or 72 hours, compared to thecorresponding Nanobody of the invention per se.

In a preferred, but non-limiting aspect of the invention, suchderivatives or polypeptides may exhibit a serum half-life in human of atleast about 12 hours, preferably at least 24 hours, more preferably atleast 48 hours, even more preferably at least 72 hours or more. Forexample, such derivatives or polypeptides may have a half-life of atleast 5 days (such as about 5 to 10 days), preferably at least 9 days(such as about 9 to 14 days), more preferably at least about 10 days(such as about 10 to 15 days), or at least about 11 days (such as about11 to 16 days), more preferably at least about 12 days (such as about 12to 18 days or more), or more than 14 days (such as about 14 to 19 days).

According to one aspect of the invention the polypeptides are capable ofbinding to one or more molecules which can increase the half-life of thepolypeptide in vivo.

The polypeptides of the invention are stabilised in vivo and theirhalf-life increased by binding to molecules which resist degradationand/or clearance or sequestration. Typically, such molecules arenaturally occurring proteins which themselves have a long half-life invivo.

Another preferred, but non-limiting example of a multispecificpolypeptide of the invention comprises at least one Nanobody of theinvention and at least one Nanobody that directs the polypeptide of theinvention towards, and/or that allows the polypeptide of the inventionto penetrate or to enter into specific organs, tissues, cells, or partsor compartments of cells, and/or that allows the Nanobody to penetrateor cross a biological barrier such as a cell membrane, a cell layer suchas a layer of epithelial cells, a tumor including solid tumours, or theblood-brain-barrier. Examples of such Nanobodies include Nanobodies thatare directed towards specific cell-surface proteins, markers or epitopesof the desired organ, tissue or cell (for example cell-surface markersassociated with tumor cells), and the single-domain brain targetingantibody fragments described in WO 02/057445 and WO 06/040153, of whichFC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQ ID NO: 190 of WO06/040154) are preferred examples.

In the polypeptides of the invention, the one or more Nanobodies and theone or more polypeptides may be directly linked to each other (as forexample described in WO 99/23221) and/or may be linked to each other viaone or more suitable spacers or linkers, or any combination thereof.

Suitable spacers or linkers for use in multivalent and multispecificpolypeptides will be clear to the skilled person, and may generally beany linker or spacer used in the art to link amino acid sequences.Preferably, said linker or spacer is suitable for use in constructingproteins or polypeptides that are intended for pharmaceutical use.

Some particularly preferred spacers include the spacers and linkers thatare used in the art to link antibody fragments or antibody domains.These include the linkers mentioned in the general background art citedabove, as well as for example linkers that are used in the art toconstruct diabodies or ScFv fragments (in this respect, however, itsshould be noted that, whereas in diabodies and in ScFv fragments, thelinker sequence used should have a length, a degree of flexibility andother properties that allow the pertinent V_(H) and V_(L) domains tocome together to form the complete antigen-binding site, there is noparticular limitation on the length or the flexibility of the linkerused in the polypeptide of the invention, since each Nanobody by itselfforms a complete antigen-binding site).

For example, a linker may be a suitable amino acid sequence, and inparticular amino acid sequences of between 1 and 50, preferably between1 and 30, such as between 1 and 10 amino acid residues. Some preferredexamples of such amino acid sequences include gly-ser linkers, forexample of the type (gly_(x)ser_(y))_(z), such as (for example(gly₄ser)₃ or (gly₃ser₂)₃, as described in WO 99/42077 and the GS30,GS15, GS9 and GS7 linkers described in the applications by Ablynxmentioned herein (see for example WO 06/040153 and WO 06/122825), aswell as hinge-like regions, such as the hinge regions of naturallyoccurring heavy chain antibodies or similar sequences (such as describedin WO 94/04678).

Some other particularly preferred linkers are poly-alanine (such asAAA), as well as the linkers GS30 (SEQ ID NO: 85 in WO 06/122825) andGS9 (SEQ ID NO: 84 in WO 06/122825).

Other suitable linkers generally comprise organic compounds or polymers,in particular those suitable for use in proteins for pharmaceutical use.For instance, poly(ethyleneglycol) moieties have been used to linkantibody domains, see for example WO 04/081026.

It is encompassed within the scope of the invention that the length, thedegree of flexibility and/or other properties of the linker(s) used(although not critical, as it usually is for linkers used in ScFvfragments) may have some influence on the properties of the finalpolypeptide of the invention, including but not limited to the affinity,specificity or avidity for Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1,Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, more preferably Tie2,Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2 or Ang2, or for oneor more of the other antigens. Based on the disclosure herein, theskilled person will be able to determine the optimal linker(s) for usein a specific polypeptide of the invention, optionally after somelimited routine experiments.

For example, in multivalent polypeptides of the invention that compriseNanobodies directed against a multimeric antigen (such as a multimericreceptor or other protein), the length and flexibility of the linker arepreferably such that it allows each Nanobody of the invention present inthe polypeptide to bind to the antigenic determinant on each of thesubunits of the multimer. Similarly, in a multispecific polypeptide ofthe invention that comprises Nanobodies directed against two or moredifferent antigenic determinants on the same antigen (for exampleagainst different epitopes of an antigen and/or against differentsubunits of a multimeric receptor, channel or protein), the length andflexibility of the linker are preferably such that it allows eachNanobody to bind to its intended antigenic determinant. Again, based onthe disclosure herein, the skilled person will be able to determine theoptimal linker(s) for use in a specific polypeptide of the invention,optionally after some limited routine experiments.

It is also within the scope of the invention that the linker(s) usedconfer one or more other favourable properties or functionality to thepolypeptides of the invention, and/or provide one or more sites for theformation of derivatives and/or for the attachment of functional groups(e.g. as described herein for the derivatives of the Nanobodies of theinvention). For example, linkers containing one or more charged aminoacid residues (see Table A-2 above) can provide improved hydrophilicproperties, whereas linkers that form or contain small epitopes or tagscan be used for the purposes of detection, identification and/orpurification. Again, based on the disclosure herein, the skilled personwill be able to determine the optimal linkers for use in a specificpolypeptide of the invention, optionally after some limited routineexperiments.

Finally, when two or more linkers are used in the polypeptides of theinvention, these linkers may be the same or different. Again, based onthe disclosure herein, the skilled person will be able to determine theoptimal linkers for use in a specific polypeptide of the invention,optionally after some limited routine experiments.

Usually, for easy of expression and production, a polypeptide of theinvention will be a linear polypeptide. However, the invention in itsbroadest sense is not limited thereto. For example, when a polypeptideof the invention comprises three of more Nanobodies, it is possible tolink them by use of a linker with three or more “arms”, which each “arm”being linked to a Nanobody, so as to provide a “star-shaped” construct.It is also possible, although usually less preferred, to use circularconstructs.

The invention also comprises derivatives of the polypeptides of theinvention, which may be essentially analogous to the derivatives of theNanobodies of the invention, i.e. as described herein.

The invention also comprises proteins or polypeptides that “essentiallyconsist” of a polypeptide of the invention (in which the wording“essentially consist of” has essentially the same meaning as indicatedhereinabove).

According to one aspect of the invention, the polypeptide of theinvention is in essentially isolated from, as defined herein.

The amino acid sequences, Nanobodies, polypeptides and nucleic acids ofthe invention can be prepared in a manner known per se, as will be clearto the skilled person from the further description herein. For example,the Nanobodies and polypeptides of the invention can be prepared in anymanner known per se for the preparation of antibodies and in particularfor the preparation of antibody fragments (including but not limited to(single) domain antibodies and ScFv fragments). Some preferred, butnon-limiting methods for preparing the amino acid sequences, Nanobodies,polypeptides and nucleic acids include the methods and techniquesdescribed herein.

As will be clear to the skilled person, one particularly useful methodfor preparing an amino acid sequence, Nanobody and/or a polypeptide ofthe invention generally comprises the steps of:

-   i) the expression, in a suitable host cell or host organism (also    referred to herein as a “host of the invention”) or in another    suitable expression system of a nucleic acid that encodes said amino    acid sequence, Nanobody or polypeptide of the invention (also    referred to herein as a “nucleic acid of the invention”), optionally    followed by:-   ii) isolating and/or purifying the amino acid sequence, Nanobody or    polypeptide of the invention thus obtained.

In particular, such a method may comprise the steps of:

-   i) cultivating and/or maintaining a host of the invention under    conditions that are such that said host of the invention expresses    and/or produces at least one amino acid sequence, Nanobody and/or    polypeptide of the invention; optionally followed by:-   ii) isolating and/or purifying the amino acid sequence, Nanobody or    polypeptide of the invention thus obtained.

A nucleic acid of the invention can be in the form of single or doublestranded DNA or RNA, and is preferably in the form of double strandedDNA. For example, the nucleotide sequences of the invention may begenomic DNA, cDNA or synthetic DNA (such as DNA with a codon usage thathas been specifically adapted for expression in the intended host cellor host organism).

According to one aspect of the invention, the nucleic acid of theinvention is in essentially isolated from, as defined herein.

The nucleic acid of the invention may also be in the form of, be presentin and/or be part of a vector, such as for example a plasmid, cosmid orYAC, which again may be in essentially isolated form.

The nucleic acids of the invention can be prepared or obtained in amanner known per se, based on the information on the amino acidsequences for the polypeptides of the invention given herein, and/or canbe isolated from a suitable natural source. To provide analogs,nucleotide sequences encoding naturally occurring V_(HH) domains can forexample be subjected to site-directed mutagenesis, so at to provide anucleic acid of the invention encoding said analog. Also, as will beclear to the skilled person, to prepare a nucleic acid of the invention,also several nucleotide sequences, such as at least one nucleotidesequence encoding a Nanobody and for example nucleic acids encoding oneor more linkers can be linked together in a suitable manner.

Techniques for generating the nucleic acids of the invention will beclear to the skilled person and may for instance include, but are notlimited to, automated DNA synthesis; site-directed mutagenesis;combining two or more naturally occurring and/or synthetic sequences fortwo or more parts thereof), introduction of mutations that lead to theexpression of a truncated expression product; introduction of one ormore restriction sites (e.g. to create cassettes and/or regions that mayeasily be digested and/or ligated using suitable restriction enzymes),and/or the introduction of mutations by means of a PCR reaction usingone or more “mismatched” primers, using for example a sequence of anaturally occurring form of Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1,Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, more preferably Tie2,Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2 or Ang2 as atemplate. These and other techniques will be clear to the skilledperson, and reference is again made to the standard handbooks, such asSambrook et al. and Ausubel et al., mentioned above, as well as theExamples below.

The nucleic acid of the invention may also be in the form of, be presentin and/or be part of a genetic construct, as will be clear to the personskilled in the art. Such genetic constructs generally comprise at leastone nucleic acid of the invention that is optionally linked to one ormore elements of genetic constructs known per se, such as for exampleone or more suitable regulatory elements (such as a suitablepromoter(s), enhancer(s), terminator(s), etc.) and the further elementsof genetic constructs referred to herein. Such genetic constructscomprising at least one nucleic acid of the invention will also bereferred to herein as “genetic constructs of the invention”.

The genetic constructs of the invention may be DNA or RNA, and arepreferably double-stranded DNA. The genetic constructs of the inventionmay also be in a form suitable for transformation of the intended hostcell or host organism, in a form suitable for integration into thegenomic DNA of the intended host cell or in a form suitable forindependent replication, maintenance and/or inheritance in the intendedhost organism. For instance, the genetic constructs of the invention maybe in the form of a vector, such as for example a plasmid, cosmid, YAC,a viral vector or transposon. In particular, the vector may be anexpression vector, i.e. a vector that can provide for expression invitro and/or in vivo (e.g. in a suitable host cell, host organism and/orexpression system).

In a preferred but non-limiting aspect, a genetic construct of theinvention comprises

-   i) at least one nucleic acid of the invention; operably connected to-   ii) one or more regulatory elements, such as a promoter and    optionally a suitable terminator;    and optionally also-   iii) one or more further elements of genetic constructs known per    se;    in which the terms “regulatory element”, “promoter”, “terminator”    and “operably connected” have their usual meaning in the art (as    further described herein); and in which said “further elements”    present in the genetic constructs may for example be 3′- or 5′-UTR    sequences, leader sequences, selection markers, expression    markers/reporter genes, and/or elements that may facilitate or    increase (the efficiency of) transformation or integration. These    and other suitable elements for such genetic constructs will be    clear to the skilled person, and may for instance depend upon the    type of construct used, the intended host cell or host organism; the    manner in which the nucleotide sequences of the invention of    interest are to be expressed (e.g. via constitutive, transient or    inducible expression); and/or the transformation technique to be    used. For example, regulatory sequences, promoters and terminators    known per se for the expression and production of antibodies and    antibody fragments (including but not limited to (single) domain    antibodies and ScFv fragments) may be used in an essentially    analogous manner.

Preferably, in the genetic constructs of the invention, said at leastone nucleic acid of the invention and said regulatory elements, andoptionally said one or more further elements, are “operably linked” toeach other, by which is generally meant that they are in a functionalrelationship with each other. For instance, a promoter is considered“operably linked” to a coding sequence if said promoter is able toinitiate or otherwise control/regulate the transcription and/or theexpression of a coding sequence (in which said coding sequence should beunderstood as being “under the control of” said promotor). Generally,when two nucleotide sequences are operably linked, they will be in thesame orientation and usually also in the same reading frame. They willusually also be essentially contiguous, although this may also not berequired.

Preferably, the regulatory and further elements of the geneticconstructs of the invention are such that they are capable of providingtheir intended biological function in the intended host cell or hostorganism.

For instance, a promoter, enhancer or terminator should be “operable” inthe intended host cell or host organism, by which is meant that (forexample) said promoter should be capable of initiating or otherwisecontrolling/regulating the transcription and/or the expression of anucleotide sequence—e.g. a coding sequence—to which it is operablylinked (as defined herein).

Some particularly preferred promoters include, but are not limited to,promoters known per se for the expression in the host cells mentionedherein; and in particular promoters for the expression in the bacterialcells, such as those mentioned herein and/or those used in the Examples.

A selection marker should be such that it allows—i.e. under appropriateselection conditions—host cells and/or host organisms that have been(successfully) transformed with the nucleotide sequence of the inventionto be distinguished from host cells/organisms that have not been(successfully) transformed. Some preferred, but non-limiting examples ofsuch markers are genes that provide resistance against antibiotics (suchas kanamycin or ampicillin), genes that provide for temperatureresistance, or genes that allow the host cell or host organism to bemaintained in the absence of certain factors, compounds and/or (food)components in the medium that are essential, for survival of thenon-transformed cells or organisms.

A leader sequence should be such that—in the intended host cell or hostorganism—it allows for the desired post-translational modificationsand/or such that it directs the transcribed mRNA to a desired part ororganelle of a cell. A leader sequence may also allow for secretion ofthe expression product from said cell. As such, the leader sequence maybe any pro-, pre-, or prepro-sequence operable in the host cell or hostorganism. Leader sequences may not be required for expression in abacterial cell. For example, leader sequences known per se for theexpression and production of antibodies and antibody fragments(including but not limited to single domain antibodies and ScFvfragments) may be used in an essentially analogous manner.

An expression marker or reporter gene should be such that—in the hostcell or host organism—it allows for detection of the expression of (agene or nucleotide sequence present on) the genetic construct. Anexpression marker may optionally also allow for the localisation of theexpressed product, e.g. in a specific part or organelle of a cell and/orin (a) specific cell(s), tissue(s), organ(s) or part(s) of amulticellular organism. Such reporter genes may also be expressed as aprotein fusion with the amino acid sequence of the invention. Somepreferred, but non-limiting examples include fluorescent proteins suchas GFP.

Some preferred, but non-limiting examples of suitable promoters,terminator and further elements include those that can be used for theexpression in the host cells mentioned herein; and in particular thosethat are suitable for expression in bacterial cells, such as thosementioned herein and/or those used in the Examples below. For some(further) non-limiting examples of the promoters, selection markers,leader sequences, expression markers and further elements that may bepresent/used in the genetic constructs of the invention—such asterminators, transcriptional and/or translational enhancers and/orintegration factors—reference is made to the general handbooks such asSambrook et al. and Ausubel et al. mentioned above, as well as to theexamples that are given in WO 95/07463, WO 96/23810, WO 95/07463, WO95/21191, WO 97/11094, WO 97/42320, WO 98/06737, WO 98/21355, U.S. Pat.No. 7,207,410, U.S. Pat. No. 5,693,492 and EP 1 085 089. Other exampleswill be clear to the skilled person. Reference is also made to thegeneral background art cited above and the further references citedherein.

The genetic constructs of the invention may generally be provided bysuitably linking the nucleotide sequence(s) of the invention to the oneor more further elements described above, for example using thetechniques described in the general handbooks such as Sambrook et al.and Ausubel et al., mentioned above.

Often, the genetic constructs of the invention will be obtained byinserting a nucleotide sequence of the invention in a suitable(expression) vector known per se. Some preferred, but non-limitingexamples of suitable expression vectors are those used in the Examplesbelow, as well as those mentioned herein.

The nucleic acids of the invention and/or the genetic constructs of theinvention may be used to transform a host cell or host organism, i.e.for expression and/or production of the amino acid sequence, Nanobody orpolypeptide of the invention. Suitable hosts or host cells will be clearto the skilled person, and may for example be any suitable fungal,prokaryotic or eukaryotic cell or cell line or any suitable fungal,prokaryotic or eukaryotic organism, for example:

-   -   a bacterial strain, including but not limited to gram-negative        strains such as strains of Escherichia coli; of Proteus, for        example of Proteus mirabilis; of Pseudomonas, for example of        Pseudomonas fluorescens; and gram-positive strains such as        strains of Bacillus, for example of Bacillus subtilis or of        Bacillus brevis; of Streptomyces, for example of Streptomyces        lividans; of Staphylococcus, for example of Staphylococcus        carnosus; and of Lactococcus, for example of Lactococcus lactis;    -   a fungal cell, including but not limited to cells from species        of Trichoderma, for example from Trichoderma reesei; of        Neurospora, for example from Neurospora crassa; of Sordaria, for        example from Sordaria macrospora; of Aspergillus, for example        from Aspergillus niger or from Aspergillus sojae; or from other        filamentous fungi;    -   a yeast cell, including but not limited to cells from species of        Saccharomyces, for example of Saccharomyces cerevisiae; of        Schizosaccharomyces, for example of Schizosaccharomyces pombe;        of Pichia, for example of Pichia pastoris or of Pichia        methanolica; of Hansenula, for example of Hansenula polymorpha;        of Kluyveromyces, for example of Kluyveromyces lactis; of        Arxula, for example of Arxula adeninivorans; of Yarrowia, for        example of Yarrowia lipolytica;    -   an amphibian cell or cell line, such as Xenopus oocytes;    -   an insect-derived cell or cell line, such as cells/cell lines        derived from lepidoptera, including but not limited to        Spodoptera SF9 and Sf21 cells or cells/cell lines derived from        Drosophila, such as Schneider and Kc cells;    -   a plant or plant cell, for example in tobacco plants; and/or    -   a mammalian cell or cell line, for example a cell or cell line        derived from a human, a cell or a cell line from mammals        including but not limited to CHO-cells, BHK-cells (for example        BHK-21 cells) and human cells or cell lines such as HeLa, COS        (for example COS-7) and PER.C6 cells;        as well as all other hosts or host cells known per se for the        expression and production of antibodies and antibody fragments        (including but not limited to (single) domain antibodies and        ScFv fragments), which will be clear to the skilled person.        Reference is also made to the general background art cited        hereinabove, as well as to for example WO 94/29457; WO 96/34103;        WO 99/42077; Frenken et al., (1998), supra; Riechmann and        Muyldermans, (1999), supra; van der Linden, (2000), supra;        Thomassen et al., (2002), supra; Joosten et al. (2003), supra;        Joosten et al., (2005), supra; and the further references cited        herein.

The amino acid sequences, Nanobodies and polypeptides of the inventioncan also be introduced and expressed in one or more cells, tissues ororgans of a multicellular organism, for example for prophylactic and/ortherapeutic purposes (e.g. as a gene therapy). For this purpose, thenucleotide sequences of the invention may be introduced into the cellsor tissues in any suitable way, for example as such (e.g. usingliposomes) or after they have been inserted into a suitable gene therapyvector (for example derived from retroviruses such as adenovirus, orparvoviruses such as adeno-associated virus). As will also be clear tothe skilled person, such gene therapy may be performed in vivo and/or insitu in the body of a patient by administering a nucleic acid of theinvention or a suitable gene therapy vector encoding the same to thepatient or to specific cells or a specific tissue or organ of thepatient; or suitable cells (often taken from the body of the patient tobe treated, such as explanted lymphocytes, bone marrow aspirates ortissue biopsies) may be treated in vitro with a nucleotide sequence ofthe invention and then be suitably (re-)introduced into the body of thepatient. All this can be performed using gene therapy vectors,techniques and delivery systems which are well known to the skilledperson, and for example described in Culver, K. W., “Gene Therapy”,1994, p. xii, Mary Ann Liebert, Inc., Publishers, New York, N.Y.);Giordano, Nature F Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79(1996), 911-919; Anderson, Science 256 (1992), 808-813; Verma, Nature389 (1994), 239; Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ.Res. 77 (1995) 0077-1086; Onodera, Blood 91; (1998), 30-36; Verma, GeneTher. 5 (1998), 692-699; Nabel, Ann. N.Y. Acad. Sci.: 811 (1997),289-292; Verzeletti, Hum. Gene Ther. 9 (1998), 2243-51; Wang, NatureMedicine 2 (1996), 714-716; WO 94/29469; WO 97/00957, U.S. Pat. No.5,580,859; U.S. Pat. No. 5,589,5466; or Schaper, Current Opinion inBiotechnology 7 (1996), 635-640. For example, in situ expression of ScFvfragments (Afanasieva et al., Gene Ther., 10, 1850-1859 (2003)) and ofdiabodies (Blanco et al., J. Immunol, 171, 1070-1077 (2003)) has beendescribed in the art.

For expression of the Nanobodies in a cell, they may also be expressedas so-called “intrabodies”, as for example described in WO 94/02610, WO95/22618 and U.S. Pat. No. 7,004,940; WO 03/014960; in Cattaneo, A. &Biocca, S. (1997) Intracellular Antibodies: Development andApplications. Landes and Springer-Verlag; and in Kontermann, Methods 34,(2004), 163-170.

The amino acid sequences, Nanobodies and polypeptides of the inventioncan for example also be produced in the milk of transgenic mammals, forexample in the milk of rabbits, cows, goats or sheep (see for exampleU.S. Pat. No. 6,741,957, U.S. Pat. No. 6,304,489 and U.S. Pat. No.6,849,992 for general techniques for introducing transgenes intomammals), in plants or parts of plants including but not limited totheir leaves, flowers, fruits, seed, roots or turbers (for example intobacco, maize, soybean or alfalfa) or in for example pupae of thesilkworm Bombix mori.

Furthermore, the amino acid sequences, Nanobodies and polypeptides ofthe invention can also be expressed and/or produced in cell-freeexpression systems, and suitable examples of such systems will be clearto the skilled person. Some preferred, but non-limiting examples includeexpression in the wheat germ system; in rabbit reticulocyte lysates; orin the E. coli Zubay system.

As mentioned above, one of the advantages of the use of Nanobodies isthat the polypeptides based thereon can be prepared through expressionin a suitable bacterial system, and suitable bacterial expressionsystems, vectors, host cells, regulatory elements, etc., will be clearto the skilled person, for example from the references cited above. Itshould however be noted that the invention in its broadest sense is notlimited to expression in bacterial systems.

Preferably, in the invention, an (in vivo or in vitro) expressionsystem, such as a bacterial expression system, is used that provides thepolypeptides of the invention in a form that is suitable forpharmaceutical use, and such expression systems will again be clear tothe skilled person. As also will be clear to the skilled person,polypeptides of the invention suitable for pharmaceutical use can beprepared using techniques for peptide synthesis.

For production on industrial scale, preferred heterologous hosts for the(industrial) production of Nanobodies or Nanobody-containing proteintherapeutics include strains of E. coli, Pichia pastoris, S. cerevisiaethat are suitable for large scale expression/production/fermentation,and in particular for large scale pharmaceutical (i.e. GMP grade)expression/production/fermentation. Suitable examples of such strainswill be clear to the skilled person. Such strains andproduction/expression systems are also made available by companies suchas Biovitrum (Uppsala, Sweden).

Alternatively, mammalian cell lines, in particular Chinese hamster ovary(CHO) cells, can be used for large scaleexpression/production/fermentation, and in particular for large scalepharmaceutical expression/production/fermentation. Again, suchexpression/production systems are also made available by some of thecompanies mentioned above.

The choice of the specific expression system would depend in part on therequirement for certain post-translational modifications, morespecifically glycosylation. The production of a Nanobody-containingrecombinant protein for which glycosylation is desired or required wouldnecessitate the use of mammalian expression hosts that have the abilityto glycosylate the expressed protein. In this respect, it will be clearto the skilled person that the glycosylation pattern obtained (i.e. thekind, number and position of residues attached) will depend on the cellor cell line that is used for the expression. Preferably, either a humancell or cell line is used (i.e. leading to a protein that essentiallyhas a human glycosylation pattern) or another mammalian cell line isused that can provide a glycosylation pattern that is essentially and/orfunctionally the same as human glycosylation or at least mimics humanglycosylation. Generally, prokaryotic hosts such as E. coli do not havethe ability to glycosylate proteins, and the use of lower eukaryotessuch as yeast usually leads to a glycosylation pattern that differs fromhuman glycosylation. Nevertheless, it should be understood that all theforegoing host cells and expression systems can be used in theinvention, depending on the desired amino acid sequence, Nanobody orpolypeptide to be obtained.

Thus, according to one non-limiting aspect of the invention, the aminoacid sequence, Nanobody or polypeptide of the invention is glycosylated.According to another non-limiting aspect of the invention, the aminoacid sequence, Nanobody or polypeptide of the invention isnon-glycosylated.

According to one preferred, but non-limiting aspect of the invention,the amino acid sequence, Nanobody or polypeptide of the invention isproduced in a bacterial cell, in particular a bacterial cell suitablefor large scale pharmaceutical production, such as cells of the strainsmentioned above.

According to another preferred, but non-limiting aspect of theinvention, the amino acid sequence, Nanobody or polypeptide of theinvention is produced in a yeast cell, in particular a yeast cellsuitable for large scale pharmaceutical production, such as cells of thespecies mentioned above.

According to yet another preferred, but non-limiting aspect of theinvention, the amino acid sequence, Nanobody or polypeptide of theinvention is produced in a mammalian cell, in particular in a human cellor in a cell of a human cell line, and more in particular in a humancell or in a cell of a human cell line that is suitable for large scalepharmaceutical production, such as the cell lines mentioned hereinabove.

When expression in a host cell is used to produce the amino acidsequences, Nanobodies and the polypeptides of the invention, the aminoacid sequences, Nanobodies and polypeptides of the invention can beproduced either intracellular (e.g. in the cytosol, in the periplasma orin inclusion bodies) and then isolated from the host cells andoptionally further purified; or can be produced extracellular (e.g. inthe medium in which the host cells are cultured) and then isolated fromthe culture medium and optionally further purified. When eukaryotic hostcells are used, extracellular production is usually preferred since thisconsiderably facilitates the further isolation and downstream processingof the Nanobodies and proteins obtained. Bacterial cells such as thestrains of E. coli mentioned above normally do not secrete proteinsextracellular, except for a few classes of proteins such as toxins andhaemolysin, and secretory production in E. coli refers to thetranslocation of proteins across the inner membrane to the periplasmaspace. Periplasma production provides several advantages over cytosolicproduction. For example, the N-terminal amino acid sequence of thesecreted product can be identical to the natural gene product aftercleavage of the secretion signal sequence by a specific signalpeptidase. Also, there appears to be much less protease activity in theperiplasma than in the cytoplasm. In addition, protein purification issimpler due to fewer contaminating proteins in the periplasma. Anotheradvantage is that correct disulfide bonds may form because theperiplasma provides a more oxidative environment than the cytoplasm.Proteins over expressed in E. coli are often found in insolubleaggregates, so-called inclusion bodies. These inclusion bodies may belocated in the cytosol or in the periplasm; the recovery of biologicallyactive proteins from these inclusion bodies requires adenaturation/refolding process. Many recombinant proteins, includingtherapeutic proteins, are recovered from inclusion bodies.Alternatively, as will be clear to the skilled person, recombinantstrains of bacteria that have been genetically modified so as to secretea desired protein, and in particular an amino acid sequence, Nanobody ora polypeptide of the invention, can be used.

Thus, according to one non-limiting aspect of the invention, the aminoacid sequence, Nanobody or polypeptide of the invention is an amino acidsequence, Nanobody or polypeptide that has been produced intracellularlyand that has been isolated from the host cell, and in particular from abacterial cell or from an inclusion body in a bacterial cell. Accordingto another non-limiting aspect of the invention, the amino acidsequence, Nanobody or polypeptide of the invention is an amino acidsequence, Nanobody or polypeptide that has been producedextracellularly, and that has been isolated from the medium in which thehost cell is cultivated.

Some preferred, but non-limiting promoters for use with these host cellsinclude,

-   -   for expression in E. coli: lac promoter (and derivatives thereof        such as the lacUV5 promoter); arabinose promoter; left- (PL) and        rightward (PR) promoter of phage lambda; promoter of the trp        operon; hybrid lac/trp promoters (tac and trc); T7-promoter        (more specifically that of T7-phage gene 10) and other T-phage        promoters; promoter of the Tn10 tetracycline resistance gene;        engineered variants of the above promoters that include one or        more copies of an extraneous regulatory operator sequence;    -   for expression in S. cerevisiae: constitutive: ADH1 (alcohol        dehydrogenase 1), ENO (enolase), CYC1 (cytochrome c iso-1),        GAPDH (glyceraldehydes-3-phosphate dehydrogenase), PGK1        (phosphoglycerate kinase), PYK1 (pyruvate kinase); regulated:        GAL1,10,7 (galactose metabolic enzymes), ADH2 (alcohol        dehydrogenase 2), PHOS (acid phosphatase), CUP1 (copper        metallothionein); heterologous: CaMV (cauliflower mosaic virus        ³⁵S promoter);    -   for expression in Pichia pastoris: the AOX1 promoter (alcohol        oxidase I); for expression in mammalian cells: human        cytomegalovirus (hCMV) immediate early enhancer/promoter; human        cytomegalovirus (hCMV) immediate early promoter variant that        contains two tetracycline operator sequences such that the        promoter can be regulated by the Tet repressor; Herpes Simplex        Virus thymidine kinase (TK) promoter; Rous Sarcoma Virus long        terminal repeat (RSV LTR) enhancer/promoter; elongation factor        1α (hEF-1α) promoter from human, chimpanzee, mouse or rat; the        SV40 early promoter; HIV-1 long terminal repeat promoter;        β-actin promoter;

Some preferred, but non-limiting vectors for use with these host cellsinclude:

-   -   vectors for expression in mammalian cells: pMAMneo (Clontech),        pcDNA3 (Invitrogen), pMC1neo (Stratagem), pSG5 (Stratagene),        EBO-pSV2-neo (ATCC 37593), pBPV-1 (8-2) (ATCC 37110),        pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC37199), pRSVneo        (ATCC37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460) and        1ZD35 (ATCC 37565), as well as viral-based expression systems,        such as those based on adenovirus;    -   vectors for expression in bacterial cells: pET vectors (Novagen)        and pQE vectors (Qiagen);    -   vectors for expression in yeast or other fungal cells: pYES2        (Invitrogen) and Pichia expression vectors (Invitrogen);    -   vectors for expression in insect cells: pBlueBacII (Invitrogen)        and other baculovirus vectors    -   vectors for expression in plants or plant cells: for example        vectors based on cauliflower mosaic virus or tobacco mosaic        virus, suitable strains of Agrobacterium, or Ti-plasmid based        vectors.

Some preferred, but non-limiting secretory sequences for use with thesehost cells include:

-   -   for use in bacterial cells such as E. coli; PelB, Bla, OmpA,        OmpC, OmpF, OmpT, PhoA, PhoE, MalE, Lpp, LamB, and the like; TAT        signal peptide, hemolysin C-terminal secretion signal;    -   for use in yeast: α-mating factor prepro-sequence, phosphatase        (pho1), invertase (Sue), etc.;    -   for use in mammalian cells: indigenous signal in case the target        protein is of eukaryotic origin; murine Ig κ-chain V-J2-C signal        peptide; etc.

Suitable techniques for transforming a host or host cell of theinvention will be clear to the skilled person and may depend on theintended host cell/host organism and the genetic construct to be used.Reference is again made to the handbooks and patent applicationsmentioned above.

After transformation, a step for detecting and selecting those hostcells or host organisms that have been successfully transformed with thenucleotide sequence/genetic construct of the invention may be performed.This may for instance be a selection step based on a selectable markerpresent in the genetic construct of the invention or a step involvingthe detection of the amino acid sequence of the invention, e.g. usingspecific antibodies.

The transformed host cell (which may be in the form or a stable cellline) or host organisms (which may be in the form of a stable mutantline or strain) form further aspects of the present invention.

Preferably, these host cells or host organisms are such that theyexpress, or are (at least) capable of expressing (e.g. under suitableconditions), an amino acid sequence, Nanobody or polypeptide of theinvention (and in case of a host organism: in at least one cell, part,tissue or organ thereof). The invention also includes furthergenerations, progeny and/or offspring of the host cell or host organismof the invention, that may for instance be obtained by cell division orby sexual or asexual reproduction.

To produce/obtain expression of the amino acid sequences of theinvention, the transformed host cell or transformed host organism maygenerally be kept, maintained and/or cultured under conditions such thatthe (desired) amino acid sequence, Nanobody or polypeptide of theinvention is expressed/produced. Suitable conditions will be clear tothe skilled person and will usually depend upon the host cell/hostorganism used, as well as on the regulatory elements that control theexpression of the (relevant) nucleotide sequence of the invention.Again, reference is made to the handbooks and patent applicationsmentioned above in the paragraphs on the genetic constructs of theinvention.

Generally, suitable conditions may include the use of a suitable medium,the presence of a suitable source of food and/or suitable nutrients, theuse of a suitable temperature, and optionally the presence of a suitableinducing factor or compound (e.g. when the nucleotide sequences of theinvention are under the control of an inducible promoter); all of whichmay be selected by the skilled person. Again, under such conditions, theamino acid sequences of the invention may be expressed in a constitutivemanner, in a transient manner, or only when suitably induced.

It will also be clear to the skilled person that the amino acidsequence. Nanobody or polypeptide of the invention may (first) begenerated in an immature form (as mentioned above), which may then besubjected to post-translational modification, depending on the hostcell/host organism used. Also, the amino acid sequence, Nanobody orpolypeptide of the invention may be glycosylated, again depending on thehost cell/host organism used.

The amino acid sequence, Nanobody or polypeptide of the invention maythen be isolated from the host cell/host organism and/or from the mediumin which said host cell or host organism was cultivated, using proteinisolation and/or purification techniques known per se, such as(preparative) chromatography and/or electrophoresis techniques,differential precipitation techniques, affinity techniques (e.g. using aspecific, cleavable amino acid sequence fused with the amino acidsequence, Nanobody or polypeptide of the invention) and/or preparativeimmunological techniques (i.e. using antibodies against the amino acidsequence to be isolated).

Generally, for pharmaceutical use, the polypeptides of the invention maybe formulated as a pharmaceutical preparation or compositions comprisingat least one polypeptide of the invention and at least onepharmaceutically acceptable carrier, diluent or excipient and/oradjuvant, and optionally one or more further pharmaceutically activepolypeptides and/or compounds. By means of non-limiting examples, such aformulation may be in a form suitable for oral administration, forparenteral administration (such as by intravenous, intramuscular orsubcutaneous injection or intravenous infusion), for topicaladministration, for administration by inhalation, by a skin patch, by animplant, by a suppository, etc. Such suitable administration forms—whichmay be solid, semi-solid or liquid, depending on the manner ofadministration—as well as methods and carriers for use in thepreparation thereof, will be clear to the skilled person, and arefurther described herein.

Thus, in a further aspect, the invention relates to a pharmaceuticalcomposition that contains at least one amino acid of the invention, atleast one Nanobody of the invention or at least one polypeptide of theinvention and at least one suitable carrier, diluent or excipient (i.e.suitable for pharmaceutical use), and optionally one or more furtheractive substances.

Generally, the amino acid sequences, Nanobodies and polypeptides of theinvention can be formulated and administered in any suitable mannerknown per se, for which reference is for example made to the generalbackground art cited above (and in particular to WO 04/041862, WO04/041863, WO 04/041865 and WO 04/041867) as well as to the standardhandbooks, such as Remington's Pharmaceutical Sciences, 18^(th) Ed.,Mack Publishing Company, USA (1990) or Remington, the Science andPractice of Pharmacy, 21st Edition, Lippincott Williams and Wilkins(2005).

For example, the amino acid sequences, Nanobodies and polypeptides ofthe invention may be formulated and administered in any manner known perse for conventional antibodies and antibody fragments (including ScFv'sand diabodies) and other pharmaceutically active proteins. Suchformulations and methods for preparing the same will be clear to theskilled person, and for example include preparations suitable forparenteral administration (for example intravenous, intraperitoneal,subcutaneous, intramuscular, intraluminal, intra-arterial or intrathecaladministration) or for topical (i.e. transdermal or intradermal)administration.

Preparations for parenteral administration may for example be sterilesolutions, suspensions, dispersions or emulsions that are suitable forinfusion or injection. Suitable carriers or diluents for suchpreparations for example include, without limitation, sterile water andaqueous buffers and solutions such as physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution;water oils; glycerol; ethanol; glycols such as propylene glycol or aswell as mineral oils, animal oils and vegetable oils, for example peanutoil, soybean oil, as well as suitable mixtures thereof. Usually, aqueoussolutions or suspensions will be preferred.

The amino acid sequences, Nanobodies and polypeptides of the inventioncan also be administered using gene therapy methods of delivery. See,e.g., U.S. Pat. No. 5,399,346, which is incorporated by reference in itsentirety. Using a gene therapy method of delivery, primary cellstransfected with the gene encoding an amino acid sequence, Nanobody orpolypeptide of the invention can additionally be transfected with tissuespecific promoters to target specific organs, tissue, grafts, tumors, orcells and can additionally be transfected with signal and stabilizationsequences for subcellularly localized expression.

Thus, the amino acid sequences, Nanobodies and polypeptides of theinvention may be systemically administered, e.g., orally, in combinationwith a pharmaceutically acceptable vehicle such as an inert diluent oran assimilable edible carrier. They may be enclosed in hard or softshell gelatin capsules, may be compressed into tablets, or may beincorporated directly with the food of the patient's diet. For oraltherapeutic administration, the amino acid sequences, Nanobodies andpolypeptides of the invention may be combined with one or moreexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations should contain at least 0.1% of theamino acid sequence, Nanobody or polypeptide of the invention. Theirpercentage in the compositions and preparations may, of course, bevaried and may conveniently be between about 2 to about 60% of theweight of a given unit dosage form. The amount of the amino acidsequence, Nanobody or polypeptide of the invention in suchtherapeutically useful compositions is such that an effective dosagelevel will be obtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the amino acid sequences, Nanobodies and polypeptides of theinvention, sucrose or fructose as a sweetening agent, methyl andpropylparabens as preservatives, a dye and flavoring such as cherry ororange flavor. Of course, any material used in preparing any unit dosageform should be pharmaceutically acceptable and substantially non-toxicin the amounts employed. In addition, the amino acid sequences,Nanobodies and polypeptides of the invention may be incorporated intosustained-release preparations and devices.

Preparations and formulations for oral administration may also beprovided with an enteric coating that will allow the constructs of theinvention to resist the gastric environment and pass into theintestines. More generally, preparations and formulations for oraladministration may be suitably formulated for delivery into any desiredpart of the gastrointestinal tract. In addition, suitable suppositoriesmay be used for delivery into the gastrointestinal tract.

The amino acid sequences, Nanobodies and polypeptides of the inventionmay also be administered intravenously or intraperitoneally by infusionor injection. Solutions of the amino acid sequences. Nanobodies andpolypeptides of the invention or their salts can be prepared in water,optionally mixed with a nontoxic surfactant. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, triacetin, andmixtures thereof and in oils. Under ordinary conditions of storage anduse, these preparations contain a preservative to prevent the growth ofmicro organisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form must be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the aminoacid sequences, Nanobodies and polypeptides of the invention in therequired amount in the appropriate solvent with various of the otheringredients enumerated above, as required, followed by filtersterilization. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and the freeze drying techniques, which yield a powder ofthe active ingredient plus any additional desired ingredient present inthe previously sterile-filtered solutions.

For topical administration, the amino acid sequences, Nanobodies andpolypeptides of the invention may be applied in pure form, i.e., whenthey are liquids. However, it will generally be desirable to administerthem to the skin as compositions or formulations, in combination with adermatologically acceptable carrier, which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, hydroxyalkyls or glycols or water-alcohol/glycolblends, in which the amino acid sequences, Nanobodies and polypeptidesof the invention can be dissolved or dispersed at effective levels,optionally with the aid of non-toxic surfactants. Adjuvants such asfragrances and additional antimicrobial agents can be added to optimizethe properties for a given use. The resultant liquid compositions can beapplied from absorbent pads, used to impregnate bandages and otherdressings, or sprayed onto the affected area using pump-type or aerosolsprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the amino acid sequences, Nanobodies and polypeptides of theinvention to the skin are known to the art; for example, see Jacquet etal. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith etal. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).

Useful dosages of the amino acid sequences, Nanobodies and polypeptidesof the invention can be determined by comparing their in vitro activity,and in vivo activity in animal models. Methods for the extrapolation ofeffective dosages in mice, and other animals, to humans are known to theart; for example, see U.S. Pat. No. 4,938,949.

Generally, the concentration of the amino acid sequences, Nanobodies andpolypeptides of the invention in a liquid composition, such as a lotion,will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%. Theconcentration in a semi-solid or solid composition such as a gel or apowder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.

The amount of the amino acid sequences, Nanobodies and polypeptides ofthe invention required for use in treatment will vary not only with theparticular amino acid sequence, Nanobody or polypeptide selected butalso with the route of administration, the nature of the condition beingtreated and the age and condition of the patient and will be ultimatelyat the discretion of the attendant physician or clinician. Also thedosage of the amino acid sequences, Nanobodies and polypeptides of theinvention varies depending on the target cell, tumor, tissue, graft, ororgan.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

An administration regimen could include long-term, daily treatment. By“long-term” is meant at least two weeks and preferably, several weeks,months, or years of duration. Necessary modifications in this dosagerange may be determined by one of ordinary skill in the art using onlyroutine experimentation given the teachings herein. See Remington'sPharmaceutical Sciences (Martin, E. W., ed. 4), Mack Publishing Co.,Easton, Pa. The dosage can also be adjusted by the individual physicianin the event of any complication.

In another aspect, the invention relates to a method for the preventionand/or treatment of at least one disease from the group of diseasesconsisting of diseases related to i) excessive angiogenesis such asangiogenesis such as cancer, diabetic blindness, age-related maculardegeneration, rheumatoid arthritis, psoriasis, and more than 70 otherconditions and related to ii) insufficient angiogenesis such as coronaryartery disease, stroke, and delayed wound healing, said methodcomprising administering, to a subject in need thereof, apharmaceutically active amount of an amino acid sequence of theinvention, of a Nanobody of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.

In the context of the present invention, the term “prevention and/ortreatment” not only comprises preventing and/or treating the disease,but also generally comprises preventing the onset of the disease,slowing or reversing the progress of disease, preventing or slowing theonset of one or more symptoms associated with the disease, reducingand/or alleviating one or more symptoms associated with the disease,reducing the severity and/or the duration of the disease and/or of anysymptoms associated therewith and/or preventing a further increase inthe severity of the disease and/or of any symptoms associated therewith,preventing, reducing or reversing any physiological damage caused by thedisease, and generally any pharmacological action that is beneficial tothe patient being treated.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. As will beclear to the skilled person, the subject to be treated will inparticular be a person suffering from, or at risk of, the diseases anddisorders mentioned herein.

The invention relates to a method for the prevention and/or treatment ofat least one disease or disorder that is associated with Tie1, Tie2,Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl1, Angptl3, Angptl4, Angptl5, orAngptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, morepreferably Tie2 or Ang2, with its biological or pharmacologicalactivity, and/or with the biological pathways or signalling in whichTie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4,Angptl5, or Angptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4,more preferably Tie2 or Ang2 is involved, said method comprisingadministering, to a subject in need thereof, a pharmaceutically activeamount of an amino acid sequence of the invention, of a Nanobody of theinvention, of a polypeptide of the invention, and/or of a pharmaceuticalcomposition comprising the same. In particular, the invention relates toa method for the prevention and/or treatment of at least one disease ordisorder that can be treated by modulating Tie1, Tie2, Ang1, Ang2, Ang3,Ang4, Angptl1, Angptl1, Angptl3, Angptl4, Angptl5, or Angptl6, morepreferably Tie2, Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2 orAng2, its biological or pharmacological activity, and/or the biologicalpathways or signalling in which Tie1, Tie2, Ang1, Ang2, Ang3, Ang4,Angptl1, Angptl1, Angptl3, Angptl4, Angptl5, or Angptl6, more preferablyTie2, Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2 or Ang2 isinvolved, said method comprising administering, to a subject in needthereof, a pharmaceutically active amount of an amino acid sequence ofthe invention, of a Nanobody of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.In particular, said pharmaceutically effective amount may be an amountthat is sufficient to modulate Tie1, Tie2, Ang1, Ang2, Ang3, Ang4,Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, more preferablyTie2, Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2 or Ang2, itsbiological or pharmacological activity, and/or the biological pathwaysor signalling in which Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1,Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, more preferably Tie2,Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2 or Ang2 is involved;and/or an amount that provides a level of the amino acid sequence of theinvention, of a Nanobody of the invention, of a polypeptide of theinvention in the circulation that is sufficient to modulate Tie1, Tie2,Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl5, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, morepreferably Tie2 or Ang2, its biological or pharmacological activity,and/or the biological pathways or signalling in which Tie1, Tie2, Ang1,Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, morepreferably Tie2 or Ang2 is involved.

The invention furthermore relates to a method for the prevention and/ortreatment of at least one disease or disorder that can be preventedand/or treated by administering an amino acid sequence of the invention,a Nanobody of the invention or a polypeptide of the invention to apatient, said method comprising administering, to a subject in needthereof, a pharmaceutically active amount of an amino acid sequence ofthe invention, of a Nanobody of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.

More in particular, the invention relates to a method for the preventionand/or treatment of at least one disease or disorder chosen from thegroup consisting of the diseases and disorders listed herein, saidmethod comprising administering, to a subject in need thereof, apharmaceutically active amount of an amino acid sequence of theinvention, of a Nanobody of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.

In another aspect, the invention relates to a method for immunotherapy,and in particular for passive immunotherapy, which method comprisesadministering, to a subject suffering from or at risk of the diseasesand disorders mentioned herein, a pharmaceutically active amount of anamino acid sequence of the invention, of a Nanobody of the invention, ofa polypeptide of the invention, and/or of a pharmaceutical compositioncomprising the same.

In the above methods, the amino acid sequences, Nanobodies and/orpolypeptides of the invention and/or the compositions comprising thesame can be administered in any suitable manner, depending on thespecific pharmaceutical formulation or composition to be used. Thus, theamino acid sequences, Nanobodies and/or polypeptides of the inventionand/or the compositions comprising the same can for example beadministered orally, intraperitoneally (e.g. intravenously,subcutaneously, intramuscularly, or via any other route ofadministration that circumvents the gastrointestinal tract),intranasally, transdermally, topically, by means of a suppository, byinhalation, again depending on the specific pharmaceutical formulationor composition to be used. The clinician will be able to select asuitable route of administration and a suitable pharmaceuticalformulation or composition to be used in such administration, dependingon the disease or disorder to be prevented or treated and other factorswell known to the clinician.

The amino acid sequences. Nanobodies and/or polypeptides of theinvention and/or the compositions comprising the same are administeredaccording to a regime of treatment that is suitable for preventingand/or treating the disease or disorder to be prevented or treated. Theclinician will generally be able to determine a suitable treatmentregimen, depending on factors such as the disease or disorder to beprevented or treated, the severity of the disease to be treated and/orthe severity of the symptoms thereof, the specific amino acid sequence,Nanobody or polypeptide of the invention to be used, the specific routeof administration and pharmaceutical formulation or composition to beused, the age, gender, weight, diet, general condition of the patient,and similar factors well known to the clinician.

Generally, the treatment regimen will comprise the administration of oneor more amino acid sequences, Nanobodies and/or polypeptides of theinvention, or of one or more compositions comprising the same, in one ormore pharmaceutically effective amounts or doses. The specific amount(s)or doses to administered can be determined by the clinician, again basedon the factors cited above.

Generally, for the prevention and/or treatment of the diseases anddisorders mentioned herein and depending on the specific disease ordisorder to be treated, the potency of the specific amino acid sequence,Nanobody and polypeptide of the invention to be used, the specific routeof administration and the specific pharmaceutical formulation orcomposition used, the amino acid sequences, Nanobodies and polypeptidesof the invention will generally be administered in an amount between 1gram and 0.01 microgram per kg body weight per day, preferably between0.1 gram and 0.1 microgram per kg body weight per day, such as about 1,10, 100 or 1000 microgram per kg body weight per day, eithercontinuously (e.g. by infusion), as a single daily dose or as multipledivided doses during the day. The clinician will generally be able todetermine a suitable daily dose, depending on the factors mentionedherein. It will also be clear that in specific cases, the clinician maychoose to deviate from these amounts, for example on the basis of thefactors cited above and his expert judgment. Generally, some guidance onthe amounts to be administered can be obtained from the amounts usuallyadministered for comparable conventional antibodies or antibodyfragments against the same target administered via essentially the sameroute, taking into account however differences in affinity/avidity,efficacy, biodistribution, half-life and similar factors well known tothe skilled person.

Usually, in the above method, a single amino acid sequence, Nanobody orpolypeptide of the invention will be used. It is however within thescope of the invention to use two or more amino acid sequences,Nanobodies and/or polypeptides of the invention in combination.

The Nanobodies, amino acid sequences and polypeptides of the inventionmay also be used in combination with one or more furtherpharmaceutically active compounds or principles, i.e. as a combinedtreatment regimen, which may or may not lead to a synergistic effect.Again, the clinician will be able to select such further compounds orprinciples, as well as a suitable combined treatment regimen, based onthe factors cited above and his expert judgement.

In particular, the amino acid sequences, Nanobodies and polypeptides ofthe invention may be used in combination with other pharmaceuticallyactive compounds or principles that are or can be used for theprevention and/or treatment of the diseases and disorders cited herein,as a result of which a synergistic effect may or may not be obtained.Examples of such compounds and principles, as well as routes, methodsand pharmaceutical formulations or compositions for administering themwill be clear to the clinician.

When two or more substances or principles are to be used as part of acombined treatment regimen, they can be administered via the same routeof administration or via different routes of administration, atessentially the same time or at different times (e.g. essentiallysimultaneously, consecutively, or according to an alternating regime).When the substances or principles are to be administered simultaneouslyvia the same route of administration, they may be administered asdifferent pharmaceutical formulations or compositions or part of acombined pharmaceutical formulation or composition, as will be clear tothe skilled person.

Also, when two or more active substances or principles are to be used aspart of a combined treatment regimen, each of the substances orprinciples may be administered in the same amount and according to thesame regimen as used when the compound or principle is used on its own,and such combined use may or may not lead to a synergistic effect.However, when the combined use of the two or more active substances orprinciples leads to a synergistic effect, it may also be possible toreduce the amount of one, more or all of the substances or principles tobe administered, while still achieving the desired therapeutic action.This may for example be useful for avoiding, limiting or reducing anyunwanted side-effects that are associated with the use of one or more ofthe substances or principles when they are used in their usual amounts,while still obtaining the desired pharmaceutical or therapeutic effect.

The effectiveness of the treatment regimen used according to theinvention may be determined and/or followed in any manner known per sefor the disease or disorder involved, as will be clear to the clinician.The clinician will also be able, where appropriate and on a case-by-casebasis, to change or modify a particular treatment regimen, so as toachieve the desired therapeutic effect, to avoid, limit or reduceunwanted side-effects, and/or to achieve an appropriate balance betweenachieving the desired therapeutic effect on the one hand and avoiding,limiting or reducing undesired side effects on the other hand.

Generally, the treatment regimen will be followed until the desiredtherapeutic effect is achieved and/or for as long as the desiredtherapeutic effect is to be maintained. Again, this can be determined bythe clinician.

In another aspect, the invention relates to the use of an amino acidsequence, Nanobody or polypeptide of the invention in the preparation ofa pharmaceutical composition for prevention and/or treatment of at leastone i) excessive angiogenesis such as cancer, diabetic blindness,age-related macular degeneration, rheumatoid arthritis, psoriasis, andothers, ii) insufficient angiogenesis such as coronary artery disease,stroke, and delayed wound healing; and/or for use in one or more of themethods of treatment mentioned herein.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. As will beclear to the skilled person, the subject to be treated will inparticular be a person suffering from, or at risk of, the diseases anddisorders mentioned herein.

The invention also relates to the use of an amino acid sequence,Nanobody or polypeptide of the invention in the preparation of apharmaceutical composition for the prevention and/or treatment of atleast one disease or disorder that can be prevented and/or treated byadministering an amino acid sequence, Nanobody or polypeptide of theinvention to a patient.

More in particular, the invention relates to the use of an amino acidsequence, Nanobody or polypeptide of the invention in the preparation ofa pharmaceutical composition for the prevention and/or treatment of i)excessive angiogenesis such as cancer, diabetic blindness, age-relatedmacular degeneration, rheumatoid arthritis, psoriasis, and others, andof ii) insufficient angiogenesis such as coronary artery disease,stroke, and delayed wound healing, and in particular for the preventionand treatment of one or more of the diseases and disorders listedherein.

Again, in such a pharmaceutical composition, the one or more amino acidsequences, Nanobodies or polypeptides of the invention may also besuitably combined with one or more other active principles, such asthose mentioned herein.

Finally, although the use of the Nanobodies of the invention (as definedherein) and of the polypeptides of the invention is much preferred, itwill be clear that on the basis of the description herein, the skilledperson will also be able to design and/or generate, in an analogousmanner, other amino acid sequences and in particular (single) domainantibodies against Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,Angptl3, Angptl4, Angptl5, or Angptl6, more preferably Tie2, Ang2, Ang1,Ang4, or Angptl4, more preferably Tie2 or Ang2, as well as polypeptidescomprising such (single) domain antibodies.

For example, it will also be clear to the skilled person that it may bepossible to “graft” one or more of the CDR's mentioned above for theNanobodies of the invention onto such (single) domain antibodies orother protein scaffolds, including but not limited to human scaffolds ornon-immunoglobulin scaffolds. Suitable scaffolds and techniques for suchCDR grafting will be clear to the skilled person and are well known inthe art, see for example U.S. Pat. No. 7,180,370, WO 01/27160, EP 0 605522, EP 0 460 167, U.S. Pat. No. 7,054,297, Nicaise et al., ProteinScience (2004), 13:1882-1891; Ewert et al., Methods, 2004 October;34(2):184-199; Kettleborough et al., Protein Eng. 1991 October; 4(7):773-783; O'Brien and Jones, Methods Mol. Biol. 2003: 207: 81-100;Skerra, J. Mol. Recognit. 2000: 13: 167-187, and Saerens et al., J. Mol.Biol. 2005 Sep. 23; 352(3):597-607, and the further references citedtherein. For example, techniques known per se for grafting mouse or ratCDR's onto human frameworks and scaffolds can be used in an analogousmanner to provide chimeric proteins comprising one or more of the CDR'sof the Nanobodies of the invention and one or more human frameworkregions or sequences.

It should also be noted that, when the Nanobodies of the inventionscontain one or more other CDR sequences than the preferred CDR sequencesmentioned above, these CDR sequences can be obtained in any manner knownper se, for example from Nanobodies (preferred), V_(H); domains fromconventional antibodies (and in particular from human antibodies), heavychain antibodies, conventional 4-chain antibodies (such as conventionalhuman 4-chain antibodies) or other immunoglobulin sequences directedagainst Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3,Angptl4, Angptl5, or Angptl6, more preferably Tie2, Ang2, Ang1, Ang4, orAngptl4, more preferably Tie2 or Ang2. Such immunoglobulin sequencesdirected against Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,Angptl3, Angptl4, Angptl5, or Angptl6, more preferably Tie2, Ang2, Ang1,Ang4, or Angptl4, more preferably Tie2 or Ang2 can be generated in anymanner known per se, as will be clear to the skilled person, i.e. byimmunization with Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,Angptl3, Angptl4, Angptl5, or Angptl6, more preferably Tie2, Ang2, Ang1,Ang4, or Angptl4, more preferably Tie2 or Ang2 or by screening asuitable library of immunoglobulin sequences with Tie1, Tie2, Ang1,Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, orAngptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, morepreferably Tie2 or Ang2, or any suitable combination thereof.Optionally, this may be followed by techniques such as random orsite-directed mutagenesis and/or other techniques for affinitymaturation known per se. Suitable techniques for generating suchimmunoglobulin sequences will be clear to the skilled person, and forexample include the screening techniques reviewed by Hoogenboom, NatureBiotechnology, 23, 9, 1105-1116 (2005) Other techniques for generatingimmunoglobulins against a specified target include for example theNanoclone technology (as for example described in the published USpatent application 2006-0211088), so-called SLAM technology (as forexample described in the European patent application 0 542 810), the useof transgenic mice expressing human immunoglobulins or the well-knownhybridoma techniques (see for example Larrick et al, Biotechnology, Vol.7, 1989, p. 934). All these techniques can be used to generateimmunoglobulins against Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1,Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6, more preferably Tie2,Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2 or Ang2, and theCDR's of such immunoglobulins can be used in the Nanobodies of theinvention, i.e. as outlined above. For example, the sequence of such aCDR can be determined, synthesized and/or isolated, and inserted intothe sequence of a Nanobody of the invention (e.g. so as to replace thecorresponding native CDR), all using techniques known per se such asthose described herein, or Nanobodies of the invention containing suchCDR's (or nucleic acids encoding the same) can be synthesized de novo,again using the techniques mentioned herein.

Further uses of the amino acid sequences, Nanobodies, polypeptides,nucleic acids, genetic constructs and hosts and host cells of theinvention will be clear to the skilled person based on the disclosureherein. For example, and without limitation, the amino acid sequences ofthe invention can be linked to a suitable carrier or solid support so asto provide a medium than can be used in a manner known per se to purifyTie1, Tie2, Ang1 Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4,Angptl5, or Angptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4,more preferably Tie2 or Ang2 from compositions and preparationscomprising the same. Derivatives of the amino acid sequences of theinvention that comprise a suitable detectable label can also be used asmarkers to determine (qualitatively or quantitatively) the presence ofTie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4,Angptl5, or Angptl6, more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4,more preferably Tie2 or Ang2 in a composition or preparation or as amarker to selectively detect the presence of Tie1, Tie2, Ang1, Ang2,Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, or Angptl6,more preferably Tie2, Ang2, Ang1, Ang4, or Angptl4, more preferably Tie2or Ang2 on the surface of a cell or tissue (for example, in combinationwith suitable cell sorting techniques).

The invention will now be further described by means of the followingnon-limiting figures:

FIG. 1. Tie2 binding assay for a selection of clones. Negative controlsare addition of irrelevant phage selected against a viral antigen and nophage addition.

FIG. 2. Tie2-Ang1 blocking assay of selected P.E. Negative controls areaddition of irrelevant P.E. selected against a viral antigen and no P.E.addition. 5 clones (family I, II, III and IV) show significant blockingof Ang-1 binding.

FIG. 3. Tie2-Ang1 blocking assay of purified nanobodies in a dilutionseries. Negative controls are addition of irrelevant nanobody selectedagainst a viral antigen and no nanobody addition.

FIG. 4. Tie2-Ang2 blocking assay of purified nanobodies in a dilutionseries. Negative controls are addition of irrelevant nanobody selectedagainst a viral antigen and no nanobody addition. None of the Tie2-Ang1blocking nanobodies is able to block binding of Ang2 to Tie2.

FIG. 5. Ang2 binding assay for a selection of clones. Negative controlsare addition of irrelevant phage selected against a viral antigen and nophage addition.

FIG. 6. Ang2-Tie2 blocking assay for a selection of clones. Negativecontrols are addition of irrelevant P.E. selected against a viralantigen and no P.E. addition. 6 clones (family 1) show significantblocking of Ang-2 binding to Tie2.

FIG. 7. Ang2-Tie2 blocking assay of purified nanobodies in a dilutionseries. Negative controls are addition of irrelevant nanobody selectedagainst a viral antigen and no nanobody addition.

FIG. 8. Ang1 binding assay for a selection of clones. Negative controlsare addition of irrelevant phage selected against a viral antigen and nophage addition.

FIG. 9. Ang4 binding assay for a selection of clones. Negative controlsare addition of irrelevant phage selected against a viral antigen and nophage addition.

FIG. 10. Angptl4 binding assay for a selection of clones. Negativecontrols are addition of irrelevant phage selected against a viralantigen and no phage addition.

FIGS. 11&12. Ratio of phospho-Akt to Akt (FIG. 11) and phospho-ERK toERK (FIG. 12) is reported. Ø indicate non Ang-1 stimulated samples.Among anti-Tie2 NBs tested, only Nanobody 163E9 was able to block theAng1-induced Akt and Erk phosphorylation both at 7.5 ug/ml (˜500 nM) and1 ug/ml (˜67 nM). None of the others Tie-2 Nanobodies inhibitedphosphorylation of AKt and Erk.

FIGS. 13&14. Ratio of phospho-Akt to Akt (FIG. 13) and phospho-ERK toERK (FIG. 14) is reported. Nanobody 163E9 dose-dependently inhibitedAng-1 induced phosphorylation of Akt and Erk.

FIG. 15. The Tie-2 Nanobody 163E9 reverses the anti-apoptotic effect ofAng-1

FIG. 16. Nanobody 163E9 dose-dependently inhibits Ang-1 inducedphosphorylation of Tie-2

FIG. 17. Nanobody 163E9 dose-dependently inhibits Ang-1 inducedsprouting of endothelial cells.

The invention will now be further described by means of the followingnon-limiting experimental part.

Experimental Part: Example 1 Animal Immunizations

Two llamas (161 and 166) are immunized, according to standard protocols,with 6 boosts of a cocktail 121 containing recombinant human Tie2/FcChimera (R&D Systems Cat No 313-TI, Lot No BKC06). Blood is collectedfrom these animals 5 and 8 days after boost 6. In addition,approximately 1 g of lymph node is collected from each animal 5 daysafter boost 6.

Example 2 Library Construction

Peripheral blood mononuclear cells are prepared from blood samples usingFico11-Hypaque according to the manufacturer's instructions. Next, totalRNA is extracted from these cells and lymph node tissue and used asstarting material for RT-PCR to amplify Nanobody encoding genefragments. These fragments are cloned into phagemid vector pAX50 (seebelow). Phage is prepared according to standard methods (see for examplethe prior art and applications filed by applicant cited herein).

pAX50—An expression vector is used derived from pLTC119 which containsthe LacZ promoter, a coliphage pill protein coding sequence, aresistance gene for ampicillin or carbenicillin, a multicloning site andthe gen3 leader sequence. In frame with the Nanobody® coding sequence,the vector codes for a C-terminal c-myc tag and a (His)₆ tag.

Example 3 Selections Of Phage Displaying Tie2 Binding Nanobodies

Phage libraries 161 and 166 are used for selections on recombinant humanTie2/Fc Chimera (R&D Systems Cat No 313-TI, Lot No BKC06). Tie2/Fc isimmobilized directly on. Maxisorp 96 well microtiter plates (Nunc) at 5ug/ml, 0.5 ug/ml and 0 ug/ml (control). To minimize the number of phagebinding to the Fc-portion of Tie2/Fc the phage is pre-incubated with 250ug/ml human IgG. Following incubation with the phage libraries andextensive washing, bound phage was eluted with trypsin. The eluted phageare amplified and applied in a second round of selection on 2 ug/ml, 0.2ug/ml, 0.02 ug/ml and 0 ug/ml (control) immobilized Tie2/Fc. To minimizethe number of phage binding to the Fc-portion of Tie2/Fc the phage ispre-incubated with 100 ug/ml human IgG plus 100 ug/ml rh B7.2/Fc (R&DSystems Cat No 141-B2, Lot No BOT 075031). Individual colonies obtainedfrom the eluted phage pools are grown and i) induced for new phageproduction and ii) induced with IPTG for Nanobody expression andextraction (periplasmic extracts) according to standard methods (see forexample the prior art and applications filed by applicant cited herein).

Example 4 Screening for Tie2 Binding Nanobodies

In order to determine binding specificity to Tie2, the clones are testedin an ELISA binding assay setup, using the monoclonal phage pools. Phagebinding to Tie2/Fc Chimera (R&D Systems Cat No 313-TI, Lot No BKC06) istested. Shortly, 0.2 ug/ml receptor is immobilized on Maxisorp ELISAplates (Nunc) and free binding sites are blocked using 4% Marvel skimmedmilk in PBS. Next, 10 ul of supernatant from the monoclonal phageinductions of the different clones in 100 ul 2% Marvel PBS are allowedto bind to the immobilized antigen. After incubation and a wash step,phage binding is revealed using a HRP-conjugated monoclonal-anti-M13antibody (Gentaur Cat#27942101). Binding specificity is determined basedon OD values compared to controls having received no phage and tocontrols where in a similar ELISA binding assay the same monoclonalphage are tested for binding to 0.2 ug/ml of immobilized human IgG and0.2 ug/ml of rh B7.2/Fc.

FIG. 1 and Table B-1 show a selection of clones binding to Tie2 (seeTable B-1 for definition of clones).

TABLE B-1 Nanobodies against Tie2. SEQ ID Name NO: Sequence 162-E1 455EVQLVESGGGLVQAGGSLRLSCAASGSIFSINAMGWYQQAPGKQRELVAFITSVGTTNYADSVKGRFIISRDNAKNTVYLQMNSLKPEDTAVYYCAADLHYSGPNYWGQGTQVTVSS 162-E9 456EVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREAVSCISSVDGSTHYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAAYYCAVQGYSGGYYYTCEDSADFGFWGQGTQVTVSS 162-F11 457EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYAIGWFRQAPGKEREGVACISSSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYSCSAGSVAGCIPYYWGQGTQVTVSS 162-F3 458EVQLVESGGGLVQAGDSLRLSCTTSGRTFSDDTMGWFRQAPRKEREFVAAILWDSIKTYYADSVKGRFTISRDNAKNTVYLQMDSLKPEDTAVYYCAATPTAYGTDWYRNNYHYWGQGTQVTVSS 162-H10 459EVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAVGWFRQAPGKEREGVSCIGSSYGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVQGYSGGYYYTCEDSADFGFWGQGTQVTVSS 163-E7 460EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYSMSWVRQAPGKGLEWVSAISGGGEVTTYADSVKGRFTISRDNAKNTLYLQMSSLKPEDTALYYCAEHLNFYSVSVRSSPTSQGTQVTVSS 163-E9 461EVQLVESGGGLVQPGDSLRLSCAASGFTFGSNGMRWVRQAPGKGPEWVSSINSDGTSTYYADSVKGRFTISRDNAKNTLCLQMNSLKPED TAVYYCTTTEDPYPRGQGTQVTVSS163-G8 462 EVQLVESGGGLVQPGGSLRLSCAASGFTFGSNGMRWVRQAPGKGPEWVSSINSDGTSAFYAESVKGRFTISRDNAKNTLYLQMNSLKPED TAVYYCTTTMNPNPRGQGTQVTVSS163-H8 463 EVQLVESGGGLVQPGGSLRLSCAASGFTFGSNGMRWVRQAPGKGPEWVSSINSDGTSTYYAESVKGRFTISRDNAKNTLYLQMHSLKPED TAVYYCTTTENPNPRGPGTQVTVSS

Example 5 Screening for Nanobodies Blocking Tie2-Ang1 Interaction

Clones tested positive in the Tie2 binding assay are screened for theirability to block Ang1 binding to Tie2/Fc. For this, Nanobody-containingperiplasmic extracts (P.E.) are used in an ELISA-based ligandcompetition setup. In short, 0.75 ug/ml human Ang1 (R&D Systems Cat No923-AN/CF Lot No FHWO73091) is coated in 96 well Maxisorp microtiterplates (Nunc) and blocked with 4% Marvel skimmed milk in PBS. Inparallel, 0.2 ug/ml Tie2/Fc is incubated with 10 ul of periplasmicextract P.E. containing nanobody of the different clones in 100 ul 2%Marvel/PBS. After 1 hour, the receptor-Nanobody pre-mixes are incubated1 hour with the coated ligand. Bound Tie2/Fc is detected usingHRP-conjugated goat anti-human IgG (Jackson Immunoresearch, Cat#109-035-098). Blocking activity is determined as loss of OD signal, ascompared to wells where no P.E., or irrelevant P.E., has been added.

FIG. 2 shows results of this blocking assay using a selection of clonesbinding to Tie2.

162-E1, 162-E9, 162-F11, 162-H10, 163-E7 (see Table 13-2 below) showsignificant blocking of Ang1 binding to Tie2.

TABLE B-2Nanobodies against Tie2 and able to block Ang1 binding to Tie2. SEQ IDName NO: Sequence 162-E1 455 EVQLVESGGGLVQAGGSLRLSCAASGSIFSINAMGWYQQAPGKQRELVAFITSVGTTNYADSVKGRIISRDNAKNTVYLQMNSLKPEDTAVYYCAADLHYSGPNYWGQGTQVTVSS 162-E9 456EVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREAVSCISSVDGSTHYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAAYYCAVQGYSGGYYYTCEDSADFGFWGQGTQVTVSS 162-F11 457EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYAIGWFRQAPGKEREGVACISSSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYSCSAGSVAGCIPYYWGQGTQVTVSS 162-H10 459EVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAVGWFRQAPGKEREGVSCIGSSYGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVQGYSGGYYYTCEDSADFGFWGQGTQVTVSS 163-E7 460EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYSMSWVRQAPGKGLEWVSAISGGGEVTTYADSVKGRFTISRDNAKNTLYLQMSSLKPEDTALYYCAEHLNFYSVSVRSSPTSQGTQVTVSS

Because above Nanobodies are able to block Ang1 binding to Tie2 they arethus considered antagonists of Tie2. Functional assay to confirm thisfunction can be found later in this experimental part.

Example 6 Determining Tie2-Ang1 Interaction Blocking Efficiency byTitration of Purified Nanobody

In order to determine the receptor blocking efficiency of clones testedpositive for Ang1 competition, a dilution series of purified Nanobodiesare tested in the ELISA-based ligand competition setup. In short, 0.75ug/ml human. Ang1 (R&D Systems Cat No 923-AN/CF Lot No FHWO73091) iscoated in 96 well Maxisorp microliter plates (Nunc) and blocked with 4%Marvel skimmed milk in PBS. In parallel, 0.2 ug/ml Tie2/Fc is incubatedwith a dilution series of purified Nanobodies. After 1 hour, thereceptor-Nanobody pre-mixes are incubated 1 hour with the coated ligand.Bound Tie2/Fc is detected using HRP-conjugated goat anti-human IgG(Jackson Immunoresearch, Cat #109-035-098). Blocking activity isdetermined as loss of OD signal, as compared to wells where no P.E., orirrelevant P.E., has been added. FIG. 3 shows the results of this assay.

Example 7 Screening for Tie2-Ang2 Blocking Among the Purified Tie2-Ang1Blocking Nanobodies

In order to investigate whether the clones tested positive for Ang1competition can also block binding of Ang2 to the receptor the perviouspurified Nanobodies are tested in a new ELISA-based ligand competitionsetup. In short, 0.75 ug/ml human Ang2 (R&D Systems Cat No 923-AN/CF) iscoated in 96 well Maxisorp microtiter plates (Nunc) and blocked with 4%Marvel skimmed milk in PBS. In parallel, 0.2 ug/ml Tie2/Fc is incubatedwith 150 nM of purified Nanobodies. After 1 hour, the receptor-Nanobodypre-mixes are incubated 1 hour with the coated ligand. Bound Tie2/Fc wasdetected using HRP-conjugated goat anti-human IgG (JacksonImmunoresearch. Cat #109-035-098). Blocking activity is determined asloss of OD signal, as compared to wells where no P.E., or irrelevantP.E., has been added. FIG. 4 shows the result of this example. None ofthe Tie2-Ang1 blocking nanobodies is able to block binding of Ang2 toTie2.

Sequences alignments of Tie2 binding Nanobodies (FRs in smallletters, CDRs in capital letters): 163-G8evqlvesggglvqpggslrlscaasgftfgSNGMRwvrqapgkgpewvsSINSDGTSAFY 163-H8evqlvesggglvqpggslrlscaasgftfgSNGMRwvrqapgkgpewvsSINSDGTSTYY 163-E9evqlvesggglvqpgdslrlscaasgftfgSNGMRwvrqapgkgpewvsSINSDGTSTYY 163-E7evqlvesggglvqpggslrlscaasgftfsDYSMSwvrqapgkglewvsAISGGGEVTTY 162-E1*evqlvesggglvqaggslrlscaasgsifsINAMGwyqqapgkqrelvaFITSVG-TTNY 162-F3evqlvesggglvqagdslrlscttsgrtfsDDTMGwfrqaprkerefvaAILWDSIKTYY 162-E9evqlvesggglvqpggslrlscaasgftldDYAIGwfrqapgkereavsCISSVDGSTHY 162-H10evqlvesggglvqpggslrlscaasgftldDYAVGwfrqapgkeregvsCIGSSYGSTYY 162-F11evqlvesggglvqaggslrlscaaagftfdDYAIGwfrqapgkeregvaCISSSDGSTYY 163-G8AESVKGrftisrdnakntlylqmnslkpedtavyycttTM-----NPN----------Pr 163-H8AESVKGrftisrdnakntlylqmhslkpedtavyycttTE-----NPN----------Pr 163-E9ADSVKGrftisrdnakntlclqmnslkpedtavyycttTE-----DPY----------Pr 163-E7ADSVKGrftisrdnakntlylqmsslkpedtalyycaeHL-----NFYSV---SVRSSPt 162-E1ADSVKGrfiisrdnakntvylqmnslkpedtavyycaa-------DLHYS-----GPNYw 162-F3ADSVKGrftisrdnakntvylqmdslkpedtavyycaaTPTAYGTDWYRN-----NYHYw 162-E9ADSVKGrftisrdnakdtvylqmnslkpedtaayycavQG--YSGGYYYTCEDSADFGFw 162-H10ADSVKGrftisrdnakntvylqmnslkpedtavvycavQG--YSGGYYYTCEDSADEGFw 162-F11ADSVKGrftissdnakntvylqmnslkpedtavyscsaGS--VAGCIPY---------Yw 163-G8gqgtqvtvss 163-H8 gpgtqvtvss 163-E9 gqgtqvtvss 163-E7 sqgtqvtvss 162-E1gqgtqvtvss 162-F3 gqgtqvtvss 162-E9 gqgtqvtvss 162-H10 gqgtqvtvss162-F11 gqgtqvtvss Members:Families of binders (one family of Nanobodies has same CDR3): members:−I 162-E1 −II 162-E9, 162-H10 −III 163-E7 −IV 162-F11 −V 162-F3 −VI163-E9, 163-G8, 163-H8 *q in FR2 of 162-E1 from an Amber stop codon

Example 8 Animal Immunizations

Two llamas (171 and 172) are immunized, according to standard protocols,with 6 boosts of a cocktail 121 containing:

Recombinant human Angiopoietin-1 (R&D Systems Cat No 923-AN/CF),

Recombinant human Angiopoietin-2 (R&D Systems Cat No 623-AN/CF).

Recombinant human. Angiopoietin-4 (R&D Systems Cat No 964-AN/CF),

Recombinant human Angiopoietin-like-4 (R&D Systems Cat No 3485-AN)

Blood is collected from these animals 8 days after boost 6.

Example 9 Library Construction

Peripheral blood mononuclear cells are prepared from blood samples usingFicoll-Hypaque according to the manufacturer's instructions. Next, totalRNA is extracted from these cells and used as starting material forRT-PCR to amplify Nanobody encoding gene fragments. These fragments arecloned into phagemid vector pAX50. Phage is prepared according tostandard methods (see for example the prior art and applications filedby applicant cited herein). Example results in phage libraries 171 (fromLlama 171) and 172 (from Llama 172).

Example 10 Selections of Phage Displaying Ang2 Binding Nanobodies

Phage libraries 171 and 172 are used for selections on recombinant humanAng2 (R&D Systems Cat No 623-AN/CF). Ang2 is immobilized directly onMaxisorp 96 well microliter plates (Nunc) at 5 ug/ml, 0.5 ug/ml and 0ug/ml (control). Following incubation with the phage libraries andextensive washing, bound phage is eluted with trypsin. The eluted phageare amplified and applied in a second round of selection on 2 ug/ml, 0.2ug/ml, 0.02 ug/ml and 0 ug/ml (control) immobilized Ang2. Individualcolonies obtained from the eluted phage pools are grown and i) inducedfor new phage production and ii) induced with IPTG for Nanobodyexpression and extraction (periplasmic extracts) according to standardmethods (see for example the prior art and applications filed byapplicant cited herein).

Example 11 Screening for Ang2 Binding Nanobodies

In order to determine binding specificity to Ang2, the clones are testedin an ELISA binding assay setup, using the monoclonal phage pools. Phagebinding to Ang2 (R&D Systems Cat No 623-AN/CF) is tested. Shortly, 0.2ug/ml Ang2 is immobilized on Maxisorp ELISA plates (Nuns) and freebinding sites are blocked using 4% Marvel skimmed milk in PBS. Next, 10ul of supernatant from the monoclonal phage inductions of the differentclones in 100 ul 2% Marvel. PBS are allowed to bind to the immobilizedantigen. After incubation and a wash step, phage binding is revealedusing a HRP-conjugated monoclonal-anti-M13 antibody (GentaurCat#27942101). Binding specificity is determined based on OD valuescompared to controls having received an irrelevant phage or no phage.

FIG. 5 and Table B-3 shows a selection of clones binding to Ang2.

TABLE B-3 Nanobodies against Ang2 SEQ ID Name NO: Sequence 166-C1 464EVQLVESGGGLVQAGGSLRLSCAASGPTFGSTTIGWFRQAPGKEREGVSCISTGDGSTYYAESVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCALDQAPMWSSWSAPYEYDYWGQGTQVTVSS 166-C10 465EVQLVESGGGLVQAGGSLRLSCAASGFTFGTTTIGWFRQAPGKEREGVSCISTGDGSTNYAESVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCALDQAPMWSSWSAPYEYDYWGQGTQVTVSS 166-D7 466EVQLVESGGGLVQAGGSLRLSCAASGFTFSDTTIGWFRQAPGKEREGISCISTGDGSTYYAESVKGRFTISSDNAKNTVYLQMNSLNPEDTAVYYCALDQAPLWSTWSAPYEYDYWGQGTQVTVSS 166-F8 467EVQLVESGGGLVQAGGSLRLSCAASGFTFGTTTIGWFRQAPGKEREVVSCISTGGGSTYYTESVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCALDQAPMWSNWSAPYEYDYWGQGTQVTVSS 166-G4 468EVQLVESGGGLVQAGGSLRLSCAASGFTFSDTTIGWFRQAPGKEREGISCISTGDGSTYYAESVKGRFTISSDNAKNTVYLQMNSLNPEDTAVYYCALDQAPLWSTWSAPYEYDYWGQGT QVTVSS 166-H4 469EVQLVESGGDLVQAGGSLRLSCAASGFTFGDFTIGWFRQAPGKEREGVSCINTGDGSTNYAESVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCALDQAPMWSSWSAPYEYDYWGQGTQVTVSS 166-E12 470KVQLVESGGGLVQAGGSLRLSCAASGFTFGSTTIGWFRQAPGKEREGVSCISTGDGSTYYAESVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCALDQAPMWSSWSAPYEYDYWGQGTQVTVSS 166-D4 471EVQLVESGGGLVQAGGSLRLSCVASGRIFTNTAMGWYRQAPGKWRELVATIYSGGSTKYIDSVKGRFIISRDNTRNTVHLQMNSLKPEDT AVYYCNTVGAGSYWGQGAQVTVSS

Example 12 Screening for Nanobodies Blocking Ang2-Tie2 Interaction

Clones tested positive in the Ang2 binding assay are screened for theirability to block Ang2 binding to Tie2/Fc. For this, Nanobody-containingperiplasmic extracts (P.E.) are used in an ELISA-based ligandcompetition setup. In short, 4 ug/ml human Tie2/Fc Chimera (R&D SystemsCat No 313-TI, Lot No BKC06)) is coated in 96 well Maxisorp microliterplates (Nunc) and blocked with 4% Marvel skimmed milk in PBS. Inparallel, 0.05 ug/ml biotinylated rh Ang2 (R&D Systems Cat No BT623, LotNo BNR174091) is incubated with 10 ul of periplasmic extract containingnanobody of the different clones in 100 ul 2% Marvel/PBS. After 1 hour,the biotinylated Ang2-Nanobody pre-mixes are incubated 1 hour with thecoated receptor. Bound biotinylated. Ang2 is detected usingHRP-conjugated extravidin (SIGMA E2886-1mL, 126K4801). Blocking activityis determined as loss of OD signal, as compared to wells where no P.E.,or irrelevant P.E., has been added. FIG. 6 shows results of thisblocking assay using a selection of clones binding to Ang2.

166-D7, 166-G4, 166-114, 166-C10, 166-C1, 166-F8 (see Table B-4 below)show significant blocking of Ang2 binding to Tie2.

TABLE B-4Nanobodies against Ang2 and able to block Ang2 binding to Tie2. SEQ IDName NO: Sequence 166-C1 464EVQLVESGGGLVQAGGSLRLSCAASGFTFGSTTIGWFRQAPGKEREGVSCISTGDGSTYYAESVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCALDQAPMWSSWSAPIEYDYWGQGTQVTVSS 166-C10 465EVQLVESGGGLVQAGGSLRLSCAASGFTFGTTTIGWFRQAPGKEREGVSCISTGDGSTNYAESVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCALDQAPMWSSWSAPYEYDYWGQGTQVTVSS 166-D7 466EVQLVESGGGLVQAGGSLRLSCAASGFTFSDTTIGWFRQAPGKEREGISCISTGDGSTYYAESVKGRFTISSDNAKNTVYLQMNSLNPEDTAVYYCALDQAPLWSTWSAPYEYDYWGQGTQVTVSS 166-F8 467EVQLVESGGGLVQAGGSLRLSCAASGFTFGTTTIGWFRQAPGKEREVVSCISTGGGSTYYTESVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCALDQAPMWSNWSAPYEYDYWGQGTQVTVSS 166-G4 468EVQLVESGGGLVQAGGSLRLSCAASGFTFSDTTIGWFRQAPGKEREGISCISTGDGSTYYAESVKGRFTISSDNAKNTVYLQMNSLNPEDTAVYYCALDQAPLWSTWSAPYEYDYWGQGT QVTVSS 166-H4 469EVQLVESGGDLVQAGGSLRLSCAASGFTFGDFTIGWFRQAPGKEREGVSCINTGDGSTNYAESVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCALDQAPMWSSWSAPYEYDYWGQGTQVTVSS

Example 13 Determining Ang2-Tie2 Interaction Blocking Efficiency byTitration of Purified Nanobody

In order to determine the receptor blocking efficiency of clones testedpositive for Ang2 blocking, a dilution series of purified Nanobodies aretested in the ELISA-based ligand competition setup. In short, 4 ug/mlhuman Tie2/Fc Chimera (R&D Systems Cat No 313-TI, Lot No BKC06)) iscoated in 96 well Maxisorp microtiter plates (Nunc) and blocked with 4%Marvel skimmed milk in PBS. In parallel, 0.05 ug/ml biotinylated rh Ang2(R&D Systems Cat No BT623, Lot No BNR174091) is incubated with adilution series of purified Nanobodies. After 1 hour, the biotinylatedAng2-Nanobody pre-mixes are incubated 1 hour with the coated receptor.Bound biotinylated Ang2 is detected using HRP-conjugated extravidin(SIGMA E2886-1ML, 126K4801). Blocking activity is determined as loss ofOD signal, as compared to wells where no P.E., or irrelevant P.E. hasbeen added. FIG. 7 shows the results of this assay.

Sequences alignments of Ang2 binding Nanobodies (FRs in smallletters, CDRs in capital letters): 166-D7evqlvesggglvqaggslrlscaasgftfsDTTIGwfrqapgkeregisCISTGDGSTYY 166-G4evqlvesggglvqaggslrlscaasgftfsDTTIGwfrqapgkeregisCISTGDGSTYY 166-H4evqlvesggdlvqaggslrlscaasgftfgDFTIGwfrqapgkeregvsCINTGDGSTNY 166-E12kvqlvesggglvqaggslrlscaasgftfgSTTIGwfrqapgkeregvsCISTGDGSTYY 166-C10evqlvesggglvqaggslrlscaasgftfgTTTIGwfrqapgkeregvsCISTGDGSTNY 166-C1evqlvesggglvqaggslrlscaasgftfgSTTIGwfrqapgkeregvsCISTGDGSTYY 166-F8evqlvesggglvqaggslrlscaasgftfgTTTIGwfrqapgkerevvsCISTGGGSTYY 166-D4evqlvesggglvqaggslrlscvasgriftNTANGwyrqapqkwrelva.TIYSGGSTKY 166-H5evqlvesggglvqaggslslacvvsgrfsrINSMAwsrqvpgnarelva.SVTSGGYTNY 166-D7AESVKGrftissdnakntvylqmnslnpedtavyycalDQAPLWSTWSAPYEYDYwgqgt 166-G4AESVKGrftissdnakntvylqmnslnpedtavyycalDQAPLWSTWSAPYEYDYwgqgt 166-H4AESVKGrftissdnakntvylqmnslkpedtavyycalDQAPMWSSWSAPYEYDYwgqgt 166-E12AESVKGrftissdnakntvylqmnslkpedtavyycalDQAPMWSSWSAPYEYDYwgqgt 166-C10AESVKGrftissdnakntyylqmnslkpedtavyycalDQAPMWSSWSAPYEYDYwgqgt 166-C1AESVKGrftissdnakntvylqmnslkpedtavyycalDQAPMWSSWSAPYEYDYwgqgt 166-F8TESVKGrftissdnakntvylqmnslkpedtavyycalDQAPMWSNWSAPYEYDYwgqgt 166-D4IDSVKGrfiisrdntrntvhlqmnslkpedtavyycnt.......VGAGSY....wgqga 166-H5VDSVKGrftisrdnaknaiylqmnslksedtavyycna...RVVVRTAHGFEDNYwgqgt 166-D7qvtvss 166-G4 qvtvss 166-H4 qvtvss 166-E12 qvtvss 166-C10 qvtvss 166-C1qvtvss 166-F8 qvtvss 166-D4 qvtvss 166-H5 qvtvss Members:Families of binders (one family of Nanobodies has same CDR3): members:−I 166-D7, 166-G4, 166-H4, 166-E12, 166-C10, 166-C1, 166-F8 −II 166-D4

Example 14 Selections of Phage Displaying Ang1 Binding Nanobodies

Phage libraries 171 and 172 (Example 9) are used for selections onrecombinant human Ang1 (R&D Systems Cat No 923-AN/CF, Lot No FHWO73091).Ang1 is immobilized directly on Maxisorp 96 well microtiter plates(Nunc) at 5 ug/ml, 0.5 ug/ml and 0 ug/ml (control). Following incubationwith the phage libraries and extensive washing, bound phage is elutedwith trypsin. The eluted phage are amplified and applied in a secondround of selection on 2 ug/ml, 0.2 ug/ml, 0.02 ug/ml and 0 ug/ml(control) immobilized Ang1. In this second round, and followingincubation with the phage libraries and extensive washing, bound phageis eluted with trypsin and 100 fold excess (nM compared to coated Ang1)recombinant human Tie2/Fc Chimera (R&D Systems Cat No 313-T1, Lot NoBKC06). Individual colonies obtained from the eluted phage pools aregrown and i) induced for new phage production and ii) induced with IPTGfor Nanobody expression and extraction (periplasmic extracts) accordingto standard methods (see for example the prior art and applicationsfiled by applicant cited herein).

Example 15 Screening for Ang1 Binding Nanobodies

In order to determine binding specificity to Ang1, the clones are testedin an ELISA binding assay setup, using the monoclonal phage pools. Phagebinding to Ang1 (R&D Systems Cat No 923-AN/CF, Lot No FHWO73091) istested. Shortly, 0.2 ug/ml Ang1 is immobilized on Maxisorp ELISA plates(Nunc) and free binding sites are blocked using 4% Marvel skimmed milkin PBS. Next, 10 ul of supernatant from the monoclonal phage inductionsof the different clones in 100 ul 2% Marvel PBS are allowed to bind tothe immobilized antigen. After incubation and a wash step, phage bindingis revealed using a HRP-conjugated monoclonal-anti-M13 antibody (GentaurCat#27942101). Binding specificity is determined based on OD valuescompared to controls having received an irrelevant phage or no phage.

FIG. 8 and Table B-5 show a selection of clones binding to Ang1.

TABLE B-5 Nanobodies against Ang1 SEQ ID Name NO: Sequence 173-H9 472EVQLVESGGGLVQPGGSLRLSCAASGFTLSGNWMYWLRQAPGKGLEWISTITPRGLTAYADSVKGRFTISRDIAENTLYLQMNSLKSGDTA VYYCARDKTGERRGQGTQVTVSS184-B6 473 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYAMTWVRQAPGKGLEWVSDISWDGDITTYAASVKGRFTISRDNAKKTLYLQMNSLKPEDSAVYYCNTYGYDSGRYYSYWGQGTQVTVSS 185-H5 474EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSYISSSDGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDLSGRGDVSEYEYDYWGQGTQVTVSS

Sequences alignments of Ang1 binding Nanobodies (FRs in smallletters, CDRs in capital letters): 185-H5evqlvesggglvqpggslrlscaasgftld.YYAIGwfrqapgkeregvsYISSSDGRTY 173-H9evqlvesggglvqpggslrlscaasgftlsGNWMY.wlrqapgkglewis.TITPRGLTA 184-B6evqlvesggglvqpggslrlscaasgftfs.NYAMTwvrqapgkglewvsDISWDGDITT 185-H5YADSVNGrftisrdnakntvylqmnslkpedtavyycatDLSGRGDVSEYEYDYwgqgtq 173-H9YADSVKGrftisrdiaentlylqmnslksgdtavyycarDKTGER.........rgqgtq 184-B6YAASVKGrftisrdnakktlylqmnslkpedsavyycnt..YGYDSGRYYSY..wgqgtq 185-H5vtvss 173-H9 vtvss 184-B6 vtvss Members:Families of binders (one family of Nanobodies has same CDR3): Members:−I 173-H9 −II 184-B6 −III 185-H5

Example 16 Selections of Phage Displaying Ang4 Binding Nanobodies

Phage libraries 171 and 172 (see example 9) are used for selections onrecombinant human Angiopoietin-4 (R&D Systems Cat No 964-AN/CF). Ang4 isimmobilized directly on Maxisorp 96 well microtiter plates (Nunc) at 5ug/ml, 0.5 ug/ml and 0 ug/ml (control). Following incubation with thephage libraries and extensive washing, bound phage is eluted withtrypsin. The eluted phage are amplified and applied in a second round ofselection on 2 ug/ml, 0.2 ug/ml, 0.02 ug/ml and 0 ug/ml (control)immobilized Ang4. In this second round, and following incubation withthe phage libraries and extensive washing, bound phage is eluted withtrypsin. Individual colonies obtained from the eluted phage pools aregrown and i) induced for new phage production and ii) induced with IPTGfor Nanobody expression and extraction (periplasmic extracts) accordingto standard methods (see for example the prior art and applicationsfiled by applicant cited herein).

Example 17 Screening for Ang4 Binding Nanobodies

In order to determine binding specificity to Ang4, the clones are testedin an ELISA binding assay setup, using the monoclonal phage pools. Phagebinding to Angiopoietin-4 (R&D Systems Cat No 964-AN/CF) was tested.Shortly, 0.2 ug/ml Ang1 is immobilized on Maxisorp ELISA plates (Nuns)and free binding sites are blocked using 4% Marvel skimmed milk in PBS.Next, 10 ul of supernatant from the monoclonal phage inductions of thedifferent clones in 100 ul 2% Marvel PBS are allowed to bind to theimmobilized antigen. After incubation and a wash step, phage binding isrevealed using a HRP-conjugated monoclonal-anti-M13 antibody (GentaurCat#27942101). Binding specificity is determined based on OD valuescompared to controls having received an irrelevant phage or no phage.

FIG. 9 and Table B-6 show a selection of clones binding to Ang4.

TABLE B-6 Nanobodies against Ang4 SEQ ID Name NO: Sequence 168-A3 475EVQLVESGGGLVQPGGSLRLSCAASGFTLSGNWMYWLRQAPGKGLEWISTITPRGLTAYADSVKGRFTISRDIAENTLYLQMNSLKSGD TAVYYCARDKTGERRGQGTQVTVSS168-E5 476 EVQLVESGGGLVQPGGSLRLSCAASGFTLSSNWMYWLRQAPGKGLEWISTITPRDLTAYADSVKGRFTISRDNAENTLYLQMNSLKSE DTAVYYCAKDKAGERRGQGTQVTVSS168-G3 477 EVQLVESGGGLVQPGGSLRLSCAASGSTLDYYAIGWYRQAPGKEREWVSCISSSNYGITTYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCATNTRRKYGRLCDLNADYWGQGTQVTVSS 169-A10 478EVQLVESGGGLVQPGGSLRLSCATSGFTFSPSWMYWLRQAPGKGLEWVSTITPRGLTEYANSVKGRFTISKDNAKNTLYLQMNSLKSED TAVYYCTRDKNGPPMGQGTQVTVSS169-A12 479 EVQLVESGGGLVQPGGSLRLSCVASGSIRSIIHMGWYRQAPGNERDLVAVIIDSRTTKYSESVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNALALGTDQSSTFDSWGQGTQVTVSS 169-B12 480EVQLVESGGGLVQAGGSLRLSCAASGSIFSINAMGWYRQAPGNQRDLVAAITSGDSTKYADFVKGRFTISRDNAKNTVYLQMNSLKPE DTAVYYCAAELLGKWYWGQGTQVTVSS169-C12 481 EVQLVESGGGLVQPGGSLRLSCAASGSIRSIIHMGWYRQTPGNERDMVAVIIDSRTTKYAESVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNALALGTDQSSTFDSWGQGTQVTVSS 169-C8 482EVQLVESGGGLVQPGGSLRLSCATSGFTFSTSWMYWLRQAPGKGLEWVSTITPRGLTDYTDSVKGRFTISRDSAKNTLYLQMNSLKSED TADYYCTRDKNGPPMGQGTQVTVSS169-E12 483 EVQLVESGGGLVQAGGSLRLSCAASGSIFSINTMGWYRQAPGNQRDLVAAITNGGSTKYVDSVKGRFTISRDNAKNTVYLQMNSLKPE DTAVYYCAAESLGRWGWGQGTQVTVSS169-F11 484 EVQLVESGGGLVQPGGSLRLSCATSGFTFSTSWMYWLRQAPGKGLEWVSTITPRGLTDYTNSVKGRFTVSRDNAKNTLYLQMNSLKSE DTAVYYCTKDKNGPPMGQGTQVTVSS

Sequences alignments of Ang4 binding Nanobodies (FRs in smallletters, CDRs in capital letters): 169-F11evqlvesggglvqpggslrlscatsgftfsTSWMYwlrqapgkglewvs..TITPRGLTD 169-C8evqlvesggglvqpggslrlscatsgftfsTSWMYwlrqapgkglewvs..TITPRGLTD 169-A10evqlvesggglvqpggslrlscatsgftfsPSWMYwlrqapgkglewvs..TITPRGLTE 168-E5evqlvesggglvqpggslrlscaasgftlsSNWMYwlrqapgkglewis..TITPRDLTA 168-A3evqlvesggglvqpggslrlscaasgftlsGNWMYwlrqapgkglewis..TITPRGLTA 168-G3evqlvesggglvqpggslrlscaasgstldYYAIGwyrqapgkerewvsCISSSNYGITT 169-B12evqlvesggglvqaggslrlscaasgsifsINAMGwyrqapgnqrdlva..AITSGDSTK 169-E12evqlvesggglvqaggslrlscaasgsifsINTMGwyrqapgnqrdlva..AITNGGSTK 169-A12evqlvesggglvqpggslrlscvasgsirsIIHMGwyrqapgnerdlva..VIIDSRTTK 169-C12evqlvesggglvqpggslrlscaasgsirsIIHMGwyrqtpgnerdmva..VIIDSRTTK 169-F11YTNSVKGrftvsrdnakntlylqmnslksedtavyyctk..........DKNGPP..... 169-C8YTDSVKGrftisrdsakntly1qmnslksedtadyyctr..........DKNGPP..... 169-A10YANSVKGrftiskdnakntlylqmnslksedtavyyctr..........DKNGPP..... 168-E5YADSVKGrftisrdnaentlylqmnslksedtavyycak..........DKAGER..... 168-A3YADSVKGrftisrdiaentlylqmnslksgdtavyycar..........DKTGER..... 168-G3YADSVKGrftisrdnakntvylqmnslkpedtaiyycatNTRRKYGRLODLNADY..... 169-B12YADFVKGrftisrdnakntvylqmnslkpedtavyycaa..........ELLGKWY.... 169-E12YVDSVKGrftisrdnakntvylqmnslkpedtavyycaa..........ESLGRWG.... 169-A12YSESVKGrftisrdnakntvylqmnslkpedtavyycna..........LALGTDQQSTF 169-C12YAESVKGrftisrdnakntvylqmnslkpedtavyycna..........LALGTDQSSTF 169-F11..mgqgtqvtvss 169-C8 ..mgqgtqvtvss 169-A10 ..mgqgtqvtvss 168-E5..rgqgtqvtvss 168-A3 ..rgqgtqvtvss 168-G3 ..wgqgtqvtvss 169-B12..wgqgtqvtvss 169-E12 ..wgqgtqvtvss 169-A12 DSwgqgtqvtvss 169-C12DSwgqgtqvtvss Members:Families of binders (one family of Nanobodies has same CDR3): Members:−I 169-F11, 169-C8, 169-A10 −II 168-A3, 168-E5 −III 168-G3 −IV169-B12, 169-E12 −V 169-A12, 169-C12

Example 18 Selections of Phage Displaying Angptl4 Binding Nanobodies

Phage libraries 171 and 172 (see Example 9) are used for selections onrecombinant human Angiopoietin-like-4 (R&D Systems Cat No 3485-AN).

Angptl4 is immobilized directly on Maxisorp 96 well microtiter plates(Nunc) at 5 ug/ml, 0.5 ug/ml and 0 ug/ml (control). Following incubationwith the phage libraries and extensive washing, bound phage is elutedwith trypsin. The eluted phage are amplified and applied in a secondround of selection on 2 ug/ml, 0.2 ug/ml, 0.02 ug/ml and 0 ug/ml(control) immobilized Angptl4. In this second round, and followingincubation with the phage libraries and extensive washing, bound phageis eluted with trypsin. Individual colonies obtained from the elutedphage pools are grown and i) induced for new phage production and ii)induced with IPTG for Nanobody expression and extraction (periplasmicextracts) according to standard methods (see for example the prior artand applications filed by applicant cited herein).

Example 19 Screening for Angptl4 Binding Nanobodies

In order to determine binding specificity to Angptl4, the clones aretested in an ELISA binding assay setup, using the monoclonal phagepools. Phage binding to recombinant human Angiopoietin-like-4 (R&DSystems Cat No 3485-AN) is tested. Shortly, 0.2 ug/ml Ang1 wasimmobilized on Maxisorp ELISA plates (Nunc) and free binding sites areblocked using 4% Marvel skimmed milk in PBS. Next, 10 ul of supernatantfrom the monoclonal phage inductions of the different clones in 100 ul2% Marvel PBS are allowed to bind to the immobilized antigen. Afterincubation and a wash step, phage binding is revealed using aHRP-conjugated monoclonal-anti-M13 antibody (Gentaur Cat#27942101).Binding specificity is determined based on OD values compared tocontrols having received an irrelevant phage or no phage.

FIG. 10 and Table B-7 show a selection of clones binding to Angptl4.

TABLE B-7 Nanobodies against Angptl4 SEQ ID Name NO: Sequence 170-B1 485EVQLVESGGGLVQAGGSLRLSCAASESIFSLYVTGWYRQAPGKQRELVASITSGGSLTYADSVKGRFTISRDNAKNTVHLQMHSLKPEDTAVYFCNGRSIGVDDMPYVYWGQGTQVTVSS 170-C2 486EVQLVESGGGLVQPGGSLRLSCAASGFTFSLNAMTWVRQAPGKGLEWVSTISSGGWTTSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDMAVYYCAKGSEFNGYEVRGQGTQVTVSS 170-E2 487EVQLVESGGGLVQAGGSLRLSCAASGSISSINVMGWYRQAPGKQRDLVATITRALNTAYATSVKGRFTISRDNFTNTVYLQMNSLEPEDTAVYYCNAGGYYTNLRTGGNYWGQGTQVTVSS 170-F2 488EVQLVESGGGLVQAGGSLRLSCAASGIFIIDTMGWYRQAPGKQRELVASITPTGNTNYVDSVKGRFAISRDNNKNTMHLQMNSLKPEDTAVYYCNAVYPRYYGDDDRPPVDSWGQGTRVTVSS 170-H1 489EVQLVESGGGLAQAGGSLRLSCAASGSISSINVMGWYRQAPGKQRDLVAVITRALNTNYATSVKGRFTISRDDFKDTVYLQMNSLEPEDTAVYYCNAGGYYTNLRTGGNYWGQGTQVTVSS 171-A2 490EVQLVESGGGQVQAGDSLRLSCKASRRTISTYGMGWFRQAPGDKRDLVSSISASGASTYYVDSVKGRFTISRDNIKNTVYLQMNSLKPEDAAVYYCAAAPNGRFITMSAHVDSWGQGTQVTVSS 171-A3 491EVQLVESGGGQVQAGDSLRLSCKASRRTISTYGMGWERQAPGDKRDLVSSISASGASTYYVDSVKGRFTISRDNIKNTVYLQMNSLKPEDAAVYYCAAAPNGRFITMSTHVDYWGQGTQVTVSS 171-C4 492EVQLVESGGGLVQPGGSLRLSCAASGRTFSTFNTYSMGWFRQAPGKEREFVAAISRGGNVTPYADSVKGRFAISRDNAKNTVALQMNSLKPEDTAVYYCAASKIGIASTIRYYDYWGQGTQVTVSS 171-D2 493EVQLVESGGGLVQAGGSLRLSCAASVLTFGTYTVGWFRQAPGKEREFVSIITGSGTYNDYADSVKGRFTVSRDNAKNTVYLQMNSLKSEDTAVYYCAARHWGMFSRSENDYNYWGQGTQVTVSS 171-E2 494EVQLVESGGGLVQAGASLRLSCVDSGDTFSWYAMGWFRQQAPGKEREFVSSISGGGSNTVYADSVKGRFTVSRDRAKNTVYLQMNSLKPEDSGVYYCAADKRWGSPATSRSTHDYDFWGQGTQVTVSS 171-E4 495EVQLVESGGGLVQPGGSLRLSCAASGRTFSTFNTYSMGWFRQAPGKEREFVAAISRSGNVTPYADSVKGRFAISRDNAKNTLTLQMNSLKPEDTAVYYCAASKIGIASTIRYYDYWGQGTQVTVSS 171-F3 496EVQLVESGGGLVQTGGSLRLSCAASGRSFNLYYMGWFRQAPGREREFVAGISGSGGSTFYGDSVKGRFTISRDNLKNTMYLQMNSLKPEDTAVYYCQSSRRIITNPREYGYWGQGTQVTVSS 171-G2 497EVQLVESGGGLVQAGGSLRLSCTASGLTFSMYAMAWIRLAPGKEREVIAAIDWSGGSTFYGDSVKGRFTISRDNAKNTVYLEMNSLKPEDTAVYYCAANRRIYSSGSSLSDNSLYNFWGQGTQVTVSS 171-G4 498EVQLVESGGGLVQAGGSLRLSCVASGDTFNWYAMGWFRQQAPGKEREFVSAISGGGSNIVYVDSVKGRFTVSRDRIKNTVYLQMNSLKPEDSGVYYCAVDKRWGSPATSRSTHDYDFWGQGTQVTVSS 170-G3 499EVQLVESGGGLVQAGGSLRLSCAASETIFASAMGWYRQPPGKQRELVARITRGGSTNYAESVKGRFAISRDNADSTLYLRMNNLKPEDTAVYYCNADTIGHSSSYITYWGQGTQVTVSS 171-H2 500EVQLVESGGGLVQAGGSLRLSCAASGRPFSMYAMGWFRQAPGKEREFVTVITWSGGSTYYADSVKGRFTISKDIAKNTVYLQMNSLKPDDMAVYYCAAARRYGNLYNTNNYDYWGQGTQVTVSS 171-H4 501EVQLVESGGGQVQAGDSLRLSCKASRRTISTYGMGWFRQAPGDKRDLVSSISASGASTYYVDSVKGRFTISRDNIKNTVYLQMNSLKPEDAAVYYCAAAPNGRFITMSTHVDSWGQGTQVTVSS

Sequences alignments of Angptl4 Nanobodies (FRs in smallletters, CDRs in capital letters): 171-G4evqlvesggglvqaggslrlscvasgdtfn...WYAMGwfrqqapgkerefv.SAISGGG 171-E2evqlvesggglvqagaslrlscvdsgdtfs...WYAMGwfrqqapgkerefv.SSISGGG 170-H1evqlvesggglaqaggslrlscaasgsiss...INVMGwyr.qapgkqrdlva..VITRA 170-E2evqlvesggglvqaggslrlscaasgsiss...INVMGwyr.qapgkgrdlva..TITRA 171-H2evqlvesggglvqaggslrlscaasgrpfs...MYAMGwfr.qapgkerefvt.VITWSG 171-E4evqlvesggglvqpggslrlscaasgrtfsTFNTYSMGwfr.qapgkerefva.AISRSG 171-C4evqlvesggglvqpggslrlscaasgrtfsTFNTYSMGwfr.qapgkerefva.AISRGG 170-F2evqlvesggglvqaggslrlscaasgifii....DTMGwyr.qapgkqrelva..SITPT 170-B1evqlvesggglvqaggslrlscaasesifs...LYVTGwyr.qapgkqrelva..SITSG 171-F3evqlvesggglvqtggslrlscaasgrsfn...LYYMGwfr.qapgrerefva.GISGSG 171-H4evqlvesgggqvqagdslrlsckasrrtis...TYGMGwfr.qapgdkrdlvs.SISASG 171-A2evqlvesgggqvqagdslrlsckasrrtis...TYGMGwfr.qapgdkrdlvs.SISASG 171-A3evqlvesgggqvqagdslrlsckasrrtis...TYGMGwfr.qapgdkrdlvs.SISASG 171-D2evqlvesggglvqaggslrlscaasvltfg...TYTVGwfr.qapgkerefvs.IITGSG 170-G3evqlvesggglvqaggslrlscaasetifa....SAMGwyr.qppgkqrelva..RITRG 170-C2evqlvesggglvqpggslrlscaasgftfs...LNAMTwvr.qapgkglewvs.TISSGG 171-G2evqlvesggglvqaggslrlsctasgltfs...MYAMAwir.lapgkerevia.AIDWSG 171-G4SNIVYVDSVKGrftvsrdrikntvylqmnslkpedsgvyycav...DKRWGSPATSRSTH 171-E2SNTVYADSVKGrftvsrdrakntvylqmnslkpedsgvyycaa...DKRWGSPATSRSTH 170-H1LNTNYATSVKGrftisrddfkdtvylqmnslepedtavyycna......GGYYTNLRTGG 170-E2LNTAYATSVKGrftisrdnftntvylqmnslepedtavyycna......GGYYTNLRTGG 171-H2GSTYYADSVKGrftiskdiakntvylqmnslkpddmavyycaa......ARRYGNLYNTN 171-E4NVTPYADSVKGrfaisrdnakntltlqmnslkpedtavyycaa....SKIGIASTIRYYD 171-C4NVTPYADSVKGrfaisrdnakntvalqmnslkpedtavyycaa....SKIGIASTIRYYD 170-F2GNTNYVDSVKGrfaisrdnnkntmhlqmnslkpedtavyycna...VYPRYYGDDDRPPV 170-B1GSLTYADSVKGrftisrdnakntvhlqmhslkpedtavyfcng......RSIGVDDMPYV 171-F3GSTFYGDSVKGrftisrdnlkntmylqmnslkpedtavyycqs.....SRRIITNPREYG 171-H4ASTYYVDSVKGrftisrdnikntvylqmnslkpedaavyycaa....APNGRFITMSTHV 171-A2ASTYYVDSVKGrftisrdnikntvylqmnslkpedaavyycaa....APNGRFITMSAHV 171-A3ASTYYVDSVKGrftisrdnikntvylqmnslkpedaavyycaa....APNGRFITMSTHV 171-D2TYNDYADSVKGrftvsrdnakntvylqmnslksedtavyycaa....RHWGMFSRSENDY 170-G3GSTNYAESVKGrfaisrdnadstlylrmnnlkpedtavyycna.......DTIGHSSSYI 170-C2WTTSYADSVKGrftisrdnakntlylqmnslkpedmavyycak.......GSEFNGYEV. 171-G2GSTFYGDSVKGrftisrdnakntvylemnslkpedtavyycaaNRRIYSSGSSLSDNSLY 171-G4DYDFwgqgtqvtvss 171-E2 DYDFwgqgtqvtvss 170-H1 NY..wgqgtqvtvss 170-E2NY..wgqgtqvtvss 171-H2 NYDYwgqgtqvtvss 171-E4 Y...wgqgtqvtvss 171-C4Y...wgqgtqvtvss 170-F2 DS..wgqgtrvtvss 170-B1 Y...wgqgtqvtvss 171-F3Y...wgqgtqvtvss 171-H4 DS..wgqgtqvtvss 171-A2 DS..wgqgtqvtvss 171-A3DY..wgqgtqvtvss 171-D2 NY..wgqgtqvtvss 170-G3 TY..wgqgtqvtvss 170-C2....rgqgtqvtvss 171-G2 NF..wgqgtqvtvss Members:Families of binders (one family of Nanobodies has same CDR3): members:−I 171-A3, 171-A2 −II 170-E2, 170-H1 −III 171-H2 −IV 171-E2, 171-G4 −V171-E4, 171-C4 −VI 170-G3 −VII 170-B1 −VIII 170-F2 −IX 171-F3 −X 171-D2−XI 170-C2 −XII 171-G2

Example 20 List of General In Vitro, Cell-Based or In Vivo Assays

In vitro binding assays: ELISA, Biacore

In vivo binding assay: Flow cytometry

Solid-phase receptor binding and blocking assays (Onliner et al. 2004,supra): ELISA-based assays with either immobilized ligand or receptor,where inhibition of binding of receptor/ligand is determined. E.g.suitable cell-based assay for Tie2, Ang1 or/and Ang2 Nanobodies.

Receptor activation/inactivation assays (Fiedler et al., 2003, Harfoucheand Hussain, 2006: both supra): Western blot detection of phosphorylatedreceptor (activated) or phosphorylation of components of the downstreamsignalling pathways. E.g. suitable cell-based assay for Tie2, Ang1or/and Ang2 Nanobodies.

Cell proliferation assays (Onliner et al., 2004, supra): Inhibition oftumour endothelial cell (e.g. specific tumor cell lines or “general”endothelial cells such as human umbilical cord endothelial cells(HUVECs) proliferation is assayed on tumour cells stimulated with orwithout addition of the neutralizing nanobody. Cell proliferation isdetermined by counting the number of live cells by FACS analysis.

In vivo angiogenesis assay (Onliner et al. 2004, supra): Assaydetermining the effect on the tumour growth by addition of neutralizingnanobodies in xenografts studies. E.g. suitable in vivo assay for Tie2,Ang1 or/and Ang2 Nanobodies.

In vivo direct anti angiogenic effect (Onliner et al. 2004, supra):Assay determining a direct antineovascular effect in viva by rat cornealangiogenesis model. E.g. suitable in vivo assay for Tie2, Ang1 or/andAng2 Nanobodies.

Lipoprotein lipase (LPL) assay: Measurement of LPL activity using³H-oleic acid as substrate (Yoshida et al., 2002, supra). E.g. suitablein vitro assay for Angptl4.

In vivo .CAM (chick chorioallantoric membrane) assay: Assay determininginhibition or not of vascularisation by addition of Angptl4 bindingnanobodies using a CHO-Angptl4 expressing cell line (Le Jan et al.,2003, supra). E.g. suitable in vivo assay for Angptl4.

In vivo animal model studies: Assay determine the effect of injectingAngptl4 nanobodies on the lipid metabolism of transgenic miceoverexpressing h Angptl4 (Koster et al., 2005, supra). E.g. suitable invivo assay for Angptl4.

Example 21 List of Particularly Preferred Embodiments of Amino AcidSequences of the Invention

Amino acid sequence comprising e.g. 2 Nanobodies with antagonisticeffect for the same target, e.g. Tie2, either being directed against twodifferent epitopes, or being against the same epitope.

-   -   Amino acid sequence comprising a Nanobody against the Tie2        receptor and a Nanobody against angiopoietin 1.    -   Amino acid sequence comprising a Nanobody against the Tie2        receptor and a Nanobody against angiopoietin 2.    -   Amino acid sequence comprising a cytotoxic compound (e.g.        peptidic toxin, e.g. immunotoxin) and a Nanobody wherein the        said Nanobody is able to disrupt at least one of the Tie/Ang or        Angptl interactions, e.g. Ang1/Tie2 or Ang2/Tie2 interactions.        The amino acid sequences of the invention such as those        presented e.g. in SEQ ID NOs: 455 to 501 may be used for        targeting specific types of cancers.

Example 22 List of Target Proteins of the Invention (Links to Nucleicand Amino Acid Sequence)

Sequences from various Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, species founde.g. on Angptl1, Angptl2, Angptl3, Angptl4, http://www.ncbi.nlm.nih.gov/Angptl5, and Angptl6 sites/entrez Human Tie1 NM_005424 Human Tie2NM_000459 Human Ang1 NM_001146 Human Ang2 NM_001147 Human Ang3 AF074332Human Ang4 NM_015985 Human Angptl1 NM_004673 Human Angptl2 BC012368Human Angptl3 NM_014495 Human Angptl4 NM_001039667 Human Angptl5NM_178127 Human Angptl6 NM_031917

Example 23 Further Analysis of Tie-2 Nanobody 163E9 Reagents Used

Recombinant Human Angiopoietin-1 (R&D SYSTEM catolog number: 923-AN);Recombinant Human Angiopoietin-2 (R&D SYSTEM catolog number: 623-AN);Anti-total Erk p44/42 MAPK (Cell Signaling Technology catolog number49102); Anti-Phospho Erk p44/42 MAPK (Thr 202/Tyr 204) (E10) (CellSignaling Technology catolog number #9106S); Anti-total Akt (C67E7)(Cell Signaling Technology catolog number 44691); Anti-Phospho-Akt(Ser473) (D9E) (Cell Signaling Technology catolog number #4060); AntiTie-2/TEK, clone Ab33 (UPSTATE catalog number #05-584);Anti-Phosphotyrosine, 4G10 (Platinum Millipore)

Example 23a The Tie-2 Nanobody 163E9 Inhibits Ang-1 InducedPhosphorylation of Akt and Erk as Determined by Bioplex Analysis

To identify Tie-2 Nanobodies that inhibits Ang-1-induced activation ofTie-2, signalling pathways, the Bio-Plex phosphoprotein and total targetassays was used. With this assay the phosphorylation and expression ofproteins in lysates derived from cell culture or tissue samples,respectively are determined. The Bio-Plex total target assay reports theabundance of the target protein in one well, while the Bio-Plexphosphoprotein assay reports the phosphorylation level of the sameprotein in a separate well.

Method:

The Bio-Plex assay used a selection of beads with different spectraladdresses, each coupled to antibodies against a different target, (intotal target assay Akt and ERK 1/2; in phosphoprotein assay Akt (Ser⁴⁷³)and ERK 1/2 (Thr²⁰²/Tyr²⁰⁴, Thr¹⁸⁵/Tyr¹⁸⁷)) The coup beads were added towells of a 96-well plate. Cell lysates, in a protein range concentrationof 200-900 μg/ml derived from HUVECs appropriately treated, were addedto the wells containing coupled beads. The incubation was left for 15-18hr. Biotin-labeled detection antibodies specific for secondary epitopeson each target are added to wells. The incubation was left for 30 min.Fluorescently labeled streptavidin reporter, able to bind tobiotin-labeled detection antibodies, was added to the wells. Theincubation was left for 10 min. After rinse, the complex was resuspendedin assay buffer. In The Bio-Flex array reader, a red classificationlaser and a green reporter laser illuminated individual beads toidentify each bead's spectral address and associated reporter signal.Dyed beads were identified by their internal fluorescent signature, thelevel of target bound to beads was indicated by intensity of reportersignal. Multiplex data were reported simultaneously.

HUVECs (Human umbilical vein endothelial cells) were obtained bytreating human umbilical cord vein with collagenase and cultured in M199containing 20% FCS (2% Penicillin-Streptomycin, brain extract and 25 μgHeparin sodium sulfate). After starvation for 3-4 hr in M199 containing0.5% BSA, the cells were treated with indicated concentration of Tie-2Nanobodies for 10 min and then stimulated with 100 ng/ml h-Ang-1 for 10min. Cells were rinsed in ice-cold cell wash buffer and lysed in bufferwith protease and phosphatase inhibitors. Proteins concentration weremeasured through BCA (Bicinchoninic acid) assay and an equal amount ofprotein for each sample, ranging between 200-900 μg/ml, was used forBio-plex analysis. Ratio of phospho-Akt to Akt and phospho-ERK to ERK isreported in FIGS. 11 and 12 respectively. Ø indicate non Ang-1stimulated samples. Among anti-Tie2 NBs tested, only Nanobody 163E9 wasable to block the Ang1-induced Akt and Erk phosphorylation both at 7.5ug/ml (−500 nM) and 1 ug/ml (−67 nM). None of the others Tie-2Nanobodies inhibited phosphorylation of AKt and Erk.

Example 23b Nanobody 163E9 Dose-Dependently Inhibits Ang-1 InducedPhosphorylation of Akt and Erk as Determined by Western Blotting

HUVEC (Human umbilical vein endothelial cells) were obtained by treatinghuman umbilical cord vein with collagenase and cultured in M199containing 20% FCS (2% Penicillin-Streptomycin, brain extract and 25 μgHeparin sodium sulfate).

HUVECs were plated in 6-well plates and used in subconfluent condition(1.5±10⁵/9.6 mm dishes). After starvation in M199 containing 0.5% BSAfor 3-4 hr, the cells were treated with indicated concentration (ofNanobodies for 10 min and then stimulated with 100 ng/ml h-Ang-1 for 10mM. Cells were rinsed in ice-cold PBS and lysed in boiling buffer (500mM Tris HCl, ph 6.8; 10% SDS, Glycerol). Lysates were clarified bycentrifugation and proteins concentration was measured through BCA(Bicinchoninic acid) assay. 10 μg proteins were resolved by 10%SDS-PAGE, transferred to nitrocellulose membrane and subject to WesternBlot analysis with anti-total Erk 1/2, anti-phospho-Erk 1/2, anti totalAkt and anti-phospho-Akt. The corresponding chemiluminescent signal isacquired and quantified by a CCD camera. Ratio of phospho-Akt to Akt(FIG. 13) and phospho-ERK to ERK (FIG. 14) is reported. Nanobody 163E9dose-dependently inhibited Ang-1 induced phosphorylation of Akt and Erk.

Example 23c The Tie-2 Nanobody 163E9 Reverses the Anti-Apoptotic Effectof Ang-1

Serum starvation of HUVECS is known to result in apoptotic cell death, aprocess that can be inhibited by Ang-1. To further demonstrate that theTie-2 Nanobody 163E9 interferes with Ang-1 induced activities throughTie-2, it was investigated if Nanobody 163E9 would be able to reversethe anti-apoptotic activity of Ang-1.

Apoptosis experiments were performed using the Cell Death DetectionELISA^(PLUS) kit (Roche) evaluating the level of nucleosome associatedDNA fragments. HUVECs cells were seeded in 24 wells (2 10⁴ cells/well)or 6 wells (9.8 10⁴ cells/well) and treated over-night with differentgrowth factors indicated. Buffers and reagents used in the procedure aresupplied with the kit. Cells were lysed with 200 μl or 980 μl of LysisBuffer for 30 min at room temperature and lysates were centrifuged at200 g for 10 min. ELISA assay was performed with 20 μl of the samplesupernatant and 80 μl of the immunoreagent. The immunoreagent wasprepared by mixing 1/20 volume of Anti-DNA-HRP and 1/20Anti-histon-biotin with 18/20 volumes of incubation Buffer. Theimmunoassay binding reaction was allowed to proceed for 2 hours afterwhich the excess of reagent was removed with two washes of IncubationBuffer (200 μl each). The quantitative determination of the amount ofnucleosome was assessed by the evaluation of HRP (Horse ReadishPeroxidase) retained by the immunocomplex which is photometricallymeasured with ABTS as substrate. Finally the colorimetric reaction isblocked after 10-15 min with ABST Stop Solution.

Nanobodies against Tie-2 were tested in HUVECs cells, stimulatedover-night with Ang-1 (300 ng/ml) or growth factors-starved (SF) as acontrol. As shown in FIG. 15, Ang-1 strongly inhibited apoptosisfollowing serum starvation. Importantly, Nanobody 163E9 dose-dependentlyinhibited the anti-apoptotic activity of Ang-1. Indeed lowering theNanobody concentrations resulted in reduced cell apoptotsis.

Example 23d Nanobody 163E9 Dose-Dependently Inhibits Ang-1 InducedPhosphorylation of Tie-2

Following binding of Ang-1 to Tie-2, the cytoplasmic tail of Tie-2becomes phosphorylated. To further demonstrate that the Tie-2 Nanobody163E9 interferes with Ang-1-induced activities through Tie-2, it wasinvestigated if Nanobody 163E9 would be able to inhibit phosphorylationof Tie-2.

HUVEC (Human umbilical vein endothelial cells) were obtained by treatinghuman umbilical cord vein with collagenase and cultured in M199containing 20% FCS (2% Penicillin-Streptomycin, brain extract and 25 μgHeparin sodium sulfate).

After starvation in M199 containing 0.5% BSA for 3-4 hr, the cells weretreated with indicated concentration (ng/ml) of Nanobodies for 10 minand then stimulated with h-Ang-1 for 10 min. Cells were rinsed inice-cold PBS1× and lysed at 4° C. in EB buffer (10 mM TrisHCl, ph 7.5,150 mM NaCl, 5 mM EDTA, 1% Triton X100, 10% Glycerol) with protease andphosphatase inhibitors (50 μg/ml pepstatin, 50 μg/ml leupeptin, 10 μg/mlaprotinin, 1 mM phenylmethylsulfonyl fluoride, 100 μM ZnCl₂, 1 mMNa₃VO₄). Lysates (450-800 μg) were incubated with protein G-Sepharoseand anti-Tie-2 antibody (1 μg) for 2 hr at 4° C. After washes,immunoprecipitates were resolved in 6% SDS-PAGE and immunoblotted forP-Tyr and Tie-2. As shown in FIG. 16, at the highest concentration ofNanobody 163E9 used, phosphorylation of Tie-2 was indeed reduced.

Example 23e Nanobody 163E9 Dose-Dependently Inhibits Ang-1 InducedSprouting of Endothelial Cells

HUVECS were trypsinized, counted, and suspended at a density of 4cells/μl in culture medium containing 20% Methocel (Sigma) (20 ml ofMethocel stock with 80 ml of M-199 20% FCS, 0.1 mg/ml heparin, and 0.1mg/ml brain extract). 800 cells were seeded into non-adherentround-bottom 96-well plates, and cultured overnight at 37° C. Thefollowing day the formed spheroids were harvested, centrifuged for 15′at 300 g at room temperature, and embedded into Collagen gels. A dilutedcollagen-I (Sigma, from rat tail) solution was prepared before use bymixing 7 vol collagen (equilibrated to 3 mg/ml in sterile 0.2% aceticacid pH 3, 4° C.), 1 vol 10×M-199, 1 vol 0.1 N NaOH, and 1 vol 0.2 MHEPES pH 7.3. The EC spheroids were suspended in 200 μl of M-199 mediumcontaining 40% FCS with or without 100 ng/ml Ang1 and Nanobody 163E9 atconcentrations indicated, and mixed with an equal volume of dilutedcollagen solution. The spheroids were rapidly transferred into 96-wellplates (400 μl/well) to allow polymerizing.

Capillary-like sprouts were examined with inverted-phase contrastmicroscope (Leica Microsystem, Heerbrugg, Switzerland) and photographed.The lengths and projected areas of the capillary-like structures werequantified with the imaging software winRHIZO Pro (Regent InstrumentsInc.).

As shown in FIG. 17, Nanobody 163E9 dose-dependently inhibited sproutingof HUVEC cells induced by Ang-1.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All references disclosed herein are incorporated by reference in theirentirety for the purpose and information indicated in the specification.

Preferred Embodiments

1. Amino acid sequence comprising at least one single variable domainthat is directed against and/or that specifically binds to a proteinselected from the group consisting of Tie1, Tie2, Ang1, Ang2, Ang3,Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, and Angptl 6.

2. Amino acid sequence comprising at least one single variable domainthat is directed against and/or that specifically binds to a proteinselected from the group consisting of Tie1 and Tie2.

3. Amino acid sequence comprising at least one single variable domainthat is directed against and/or that specifically binds to a proteinselected from the group consisting of Ang1, Ang2. Ang3, and Ang4.

4. Amino acid sequence comprising at least one single variable domainthat is directed against and/or that specifically binds to a proteinselected from the group consisting of Angptl1, Angptl2, Angptl3,Angptl4, Angptl5, and Angptl6.

5. Amino acid sequence according to any previous or followingembodiments that is in essentially isolated form.

6. Amino acid sequence comprising at least one single variable domainthat is directed against and/or that specifically binds to a proteinselected from the group consisting of human Tie1, human Tie2, humanAng1, human Ang2, human Ang3, human Ang4, human Angptl1, human Angptl2,human Angptl3, human Angptl4, human Angptl5, and human Angptl6.

7. Amino acid sequence comprising at least one single variable domainthat is directed against and/or that specifically binds to a proteinselected from the group consisting of human Tie1 and human Tie2.

8. Amino acid sequence comprising at least one single variable domainthat is directed against and/or that specifically binds to a proteinselected from the group consisting of human Ang1, human Ang2, humanAng3, and human Ang4.

9. Amino acid sequence comprising at least one single variable domainthat is directed against and/or that specifically binds to a proteinselected from the group consisting of human Angptl1, human Angptl2,human Angptl3, human Angptl4, human Angptl5, and human Angptl6.

10. Amino acid sequence comprising at least one single variable domainthat is directed against and/or that specifically binds to a proteinselected from the group consisting of the human Tie2, human Ang1, humanAng2, human Ang4, and human Angptl4.

11. Amino acid sequence comprising at least one single variable domainthat is directed against and/or that specifically binds to a proteinselected from the group consisting of human Tie2 and human Ang2.

12. Amino acid sequence according to any previous or followingembodiments, wherein the variable domain i) is directed against and/orspecifically binds to human Tie2; and ii) blocks the interaction betweenhuman Tie2 and at least one Ang, e.g. a human Ang.

13. Amino acid sequence according to any previous or followingembodiments, wherein the variable domain i) is directed against and/orspecifically binds to human Tie2; and ii) blocks the interaction betweenhuman Tie2 and only one Ang, e.g. a human Ang.

14. Amino acid sequence according to any previous or followingembodiments, wherein the variable domain i) is directed against and/orspecifically binds to human Tie2; and ii) blocks the interaction betweenhuman Tie2 and human Ang1.

15. Amino acid sequence according to any previous or followingembodiments, wherein the variable domain i) is directed against and/orspecifically binds to human Tie2; and ii) blocks the interaction betweenhuman Ang1 and human Tie2; and iii) does not block the interactionbetween human Ang2 and human Tie2.

16. Amino acid sequence according to any previous or followingembodiments, wherein the variable domain i) is directed against and/orspecifically binds to human Ang2; and ii) blocks the interaction betweenhuman Tie2 and human Ang2.

17. Amino acid sequence according to any previous or followingembodiments, wherein the variable domain has an antagonistic effect toat least one member of the group of proteins consisting of Tie1 andTie2.

18. An amino acid sequence according to any previous or followingembodiments, wherein the variable domain has an agonistic effect to atleast one member of the group of proteins consisting of Tie1 and Tie2.

19. Amino acid sequence according to any previous or followingembodiments, wherein the variable domain has an antagonistic effect tohuman Tie2.

20. An amino acid sequence according to any previous or followingembodiments, wherein the variable domain has an agonistic effect tohuman Tie2.

21. Amino acid sequence according to any previous or followingembodiments, wherein the variable domain is able to inhibit the assemblyof human Tie2 homodimers.

22. An amino acid sequence according to any previous or followingembodiments, wherein the variable domain is able to enhance the assemblyof human Tie2 homodimers.

23. An amino acid sequence according to any previous or followingembodiments, wherein the variable domain is able to inhibit angiogenesisas e.g. measured in any of the herein disclosed in vitro cell based—oranimal models.

24. Amino acid sequence according to any previous or followingembodiments that can specifically bind to at least one member of thegroup of proteins consisting of Tie1, Tie2, Ang1, Ang2, Ang3, Ang4,Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, and Angptl6 with adissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or less, andpreferably 10⁻⁷ to 10⁻¹² moles/liter or less and more preferably 10⁻⁸ to10⁻¹² moles/liter.

25. Amino acid sequence according to any previous or followingembodiments, that can specifically bind to at least one member of thegroup of proteins consisting of Tie1, Tie2, Ang1, Ang2, Ang3, Ang4,Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, and Angptl6 with a rate ofassociation (L-rate) of between 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹,preferably between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more preferably between10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between 10⁵ and 10⁷

26. Amino acid sequence according to any previous or followingembodiments, that can specifically bind to at least one member of thegroup of proteins consisting of Tie1, Tie2, Ang1, Ang2, Ang3, Ang4,Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, and Angptl6 with a rate ofdissociation (k_(off) rate) between 1s⁻¹ and 10⁻⁶ s⁻¹, preferablybetween 10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶s⁻¹, such as between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

27. Amino acid sequence according to any previous or followingembodiments, that can specifically bind to at least one member of thegroup of proteins consisting of Tie1, Tie2, Ang1, Ang2, Ang3, Ang4,Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, and Angptl6 with anaffinity less than 500 nM, preferably less than 200 nM, more preferablyless than 10 nM, such as less than 500 μM.

28. Amino acid sequence according to any previous or followingembodiments, that is a naturally occurring amino acid sequence (from anysuitable species) or a synthetic or semi-synthetic amino acid sequence.

29. Amino acid sequence according to any previous or followingembodiments, that comprises an immunoglobulin fold or that undersuitable conditions is capable of forming an immunoglobulin fold.

30. Amino acid sequence according to any previous or followingembodiments, that essentially consists of 4 framework regions (FR1 toFR4 respectively) and 3 complementarity determining regions (CDR1 toCDR3 respectively).

31. Amino acid sequence according to any previous or followingembodiments that is an immunoglobulin sequence.

32. Amino acid sequence according to any previous or followingembodiments that is a naturally occurring immunoglobulin sequence (fromany suitable species) or a synthetic or semi-synthetic immunoglobulinsequence.

33. Amino acid sequence according to any previous or followingembodiments that is a humanized immunoglobulin sequence, a camelizedimmunoglobulin sequence or an immunoglobulin sequence that has beenobtained by techniques such as affinity maturation.

34. Amino acid sequence according to any previous or followingembodiments that essentially consists of a light chain variable domainsequence (e.g. a V_(L)-sequence); or of a heavy chain variable domainsequence (e.g. a V_(H)-sequence).

35. Amino acid sequence according to any previous or followingembodiments that essentially consists of a heavy chain variable domainsequence that is derived from a conventional four-chain antibody or thatessentially consist of a heavy chain variable domain sequence that isderived from heavy chain antibody.

36. Amino acid sequence according to any previous or following precedingembodiments that essentially consists of a domain antibody (or an aminoacid sequence that is suitable for use as a domain antibody), of asingle domain antibody (or an amino acid sequence that is suitable foruse as a single domain antibody), of a “dAb” (or an amino acid sequencethat is suitable for use as a dAb) or of a Nanobody™ (including but notlimited to a V_(HH) sequence).

37. Amino acid sequence according to any previous or followingembodiments that essentially consists of a Nanobody™.

38. Amino acid sequence according to any previous or followingembodiments that essentially consists of a Nanobody™ that

-   i) has 80% amino acid identity with at least one of the amino acid    sequences of SEQ ID NO's: 1 to 22, in which for the purposes of    determining the degree of amino acid identity, the amino acid    residues that form the CDR sequences are disregarded;    and in which:-   ii) preferably one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    A-3.

39. Amino acid sequence according to any previous or followingembodiments that essentially consists of a Nanobody™ that

-   i) has 80% amino acid identity with at least one of the amino acid    sequences of SEQ ID NO's: 455 to 501, in which for the purposes of    determining the degree of amino acid identity, the amino acid    residues that form the CDR sequences are disregarded;    and in which:-   ii) preferably one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    A-3.

40a. An amino acid sequence according to any previous or followingembodiments comprising at least one variable domain that cross-blocksthe binding of at least one of the amino acid sequences with SEQ ID NOs455 to 501 to a Tie, Ang and/or an Angptl.

40b. An amino acid sequence according to any previous or followingembodiments comprising at least one variable domain that iscross-blocked by at least one of the amino acid sequences with SEQ IDNOs 455 to 501 to a Tie, Ang and/or an Angptl.

40c. An amino acid sequence according to embodiments 40a or 40b whereinthe ability of said amino acid sequence to cross-block or to becross-blocked is detected in a Biacore assay.

40d. An amino acid sequence according to embodiments 40a or 40b whereinthe ability of said amino acid sequence to cross-block or to becross-blocked is detected in an ELISA assay.

40. Amino acid sequence according to any previous or followingembodiments that essentially consists of a humanized Nanobody™.

41. Construct to any following embodiments that comprises or essentiallyconsists of one or more amino acid sequences according to any ofembodiments 1 to 40, and optionally further comprises one or more othergroups, residues, moieties or binding units, optionally linked via oneor more linkers.

42. Construct according to any previous or following embodiments thatcomprises or essentially consists of one or more amino acid sequencesaccording to any of embodiments 1 to 40, and wherein the construct isable to inhibit angiogenesis as e.g. measured in any of the hereindisclosed in vitro cell based—or animal models.

43. Construct according to any previous or following embodiments, inwhich said one or more other groups, residues, moieties or binding unitsare amino acid sequences.

44. Construct according to any previous or following embodiments, inwhich said one or more linkers, if present, are one or more amino acidsequences.

45. Construct according to embodiments 42 to 44, in which said one ormore other groups, residues, moieties or binding units areimmunoglobulins.

46. Construct according to embodiments 42 to 45, in which said one ormore other groups, residues, moieties or binding units are chosen fromthe group consisting of domain antibodies, amino acid sequences that aresuitable for use as a domain antibody, single domain antibodies, aminoacid sequences that are suitable for use as a single domain antibody,“dAb”'s, amino acid sequences that are suitable for use as a dAb, orNanobodies.

47. Construct according to embodiments 42 to 46, in which said one ormore amino acid sequences of the invention are immunoglobulin sequences.

48. Construct according to embodiments 42 to 47, in which said one ormore amino acid sequences of the invention are chosen from the groupconsisting of domain antibodies, amino acid sequences that are suitablefor use as a domain antibody, single domain antibodies, amino acidsequences that are suitable for use as a single domain antibody,“dAb”'s, amino acid sequences that are suitable for use as a dAb, orNanobodies.

49. Construct, that comprises or essentially consists of one or moreNanobodies according to any of embodiments 42 to 48 and in which saidone or more other groups, residues, moieties or binding units areNanobodies.

50. Construct according to any of embodiments 41 following, which is amultivalent construct.

51. Construct according to any of embodiments 41 following, which is amultispecific construct.

52. Construct according to any of embodiments 29 to 38, which has anincreased half-life, compared to the corresponding amino acid sequenceaccording to any of embodiments 1 to 40 per se.

53. Construct according to embodiment 39, in which said one or moreother groups, residues, moieties or binding units provide the compoundor construct with increased half-life, compared to the correspondingamino acid sequence according to any of embodiments 1 to 40 per se.

54. Construct according to embodiment 53, in which said one or moreother groups, residues, moieties or binding units that provide thecompound or construct with increased half-life is chosen from the groupconsisting of serum proteins or fragments thereof, binding units thatcan bind to serum proteins, an Fc portion, and small proteins orpeptides that can bind to serum proteins.

55. Construct according to embodiment 54, in which said one or moreother groups, residues, moieties or binding units that provide thecompound or construct with increased half-life is chosen from the groupconsisting of human serum albumin or fragments thereof.

56. Construct according to embodiment 55, in which said one or moreother groups, residues, moieties or binding units that provides thecompound or construct with increased half-life are chosen from the groupconsisting of binding units that can bind to scrum albumin (such ashuman serum albumin) or a serum immunoglobulin (such as IgG).

57. Construct according to embodiment 56, in which said one or moreother groups, residues, moieties or binding units that provides thecompound or construct with increased half-life are chosen from the groupconsisting of domain antibodies, amino acid sequences that are suitablefor use as a domain antibody, single domain antibodies, amino acidsequences that are suitable for use as a single domain antibody,“dAb”'s, amino acid sequences that are suitable for use as a dAb, orNanobodies that can bind to serum albumin (such as human serum albumin)or a serum immunoglobulin (such as IgG).

58. Construct according to embodiment 57, in which said one or moreother groups, residues, moieties or binding units that provides thecompound or construct with increased half-life is a Nanobody that canbind to serum albumin (such as human serum albumin) or a serumimmunoglobulin (such as IgG).

59. Construct according to any of embodiments 53 to 58, that has a serumhalf-life that is at least 1.5 times, preferably at least 2 times, suchas at least 5 times, for example at least 10 times or more than 20times, greater than the half-life of the corresponding amino acidsequence according to any of embodiments 1 to 21 per se.

60. Construct according to any of embodiments 53 to 59, that has a serumhalf-life that is increased with more than 1 hours, preferably more than2 hours, more preferably more than 6 hours, such as more than 12 hours,or even more than 24, 48 or 72 hours, compared to the correspondingamino acid sequence according to any of embodiments 1 to 21 per se.

61. Construct according to any of embodiments 53 to 60, that has a serumhalf-life in human of at least about 12 hours, preferably at least 24hours, more preferably at least 48 hours, even more preferably at least72 hours or more; for example, of at least 5 days (such as about 5 to 10days), preferably at least 9 days (such as about 9 to 14 days), morepreferably at least about 10 days (such as about 10 to 15 days), or atleast about 11 days (such as about 11 to 16 days), more preferably atleast about 12 days (such as about 12 to 18 days or more), or more than14 days (such as about 14 to 19 days).

62. Construct according to any of embodiments 53 to 61 that comprises oressentially consists of two amino acid sequences according to any ofembodiments 1 to 28.

63. Construct according to embodiment 62, wherein said two amino acidsequences are directed against and/or specifically bind to same targetprotein which is selected from the group consisting of Tie1, Tie2, Ang1,Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, andAngptl6, said binding either being directed against two differentepitopes or being against the same epitope.

64. Construct according to embodiment 63, wherein said target protein isselected from the group consisting of human Tie1, human Tie2, humanAng1, human Ang2, human Ang3, human Ang4, human Angptl1, human Angptl2,human Angptl3, human Angptl4, human Angptl5, and human Angptl6.

65. Construct according to embodiment 64, wherein said first amino acidsequence is directed against and/or specifically binds to human Tie2 andwherein said second amino acid sequence is directed against and/orspecifically binds to human Ang1.

66. Construct according to embodiment 64, wherein said first amino acidsequence is directed against and/or specifically binds to human Tie2 andwherein said second amino acid sequence is directed against and/orspecifically binds to human Ang2.

67. Construct according to embodiment 64, wherein said first amino acidsequence is directed against and/or specifically binds to human Tie2 andwherein said second amino acid sequence is directed against and/orspecifically binds to human Ang4.

68. Construct according to embodiments 53 to 67, that comprises oressentially consists of one or more amino acid sequences according toany of embodiments 1 to 40, and optionally further comprises one or moretoxic groups, toxic residues, toxic moieties or toxic binding units,optionally linked via one or more linkers.

69. Construct according to embodiment 68, wherein the toxic group isselected from the group of immunotoxins.

70. Construct according to any previous and following embodiments thatcomprises at least 3 variable domains, e.g. Nanobodies.

71. Construct according to any previous and following embodiments thatcomprises at least 3 variable domains, e.g. Nanobodies, and that is ableto inhibit excessive angiogenesis and/or tumourgenesis, wherein saidbiological effect may be tested in a suitable cell based and/or animalmodel such as described herein.

72. Construct according to any previous and following embodiments thatcomprises at least 3 variable domains, e.g. Nanobodies, and that is ableto either inhibit cluster formation of Tie2 or able to cluster Tie2 butwithout inducing any or only partial biological effect or effects suchas inhibiting excessive angiogenesis and/or tumourgenesis to normalizedlevels, wherein said biological effect may be tested in a suitable cellbased and/or animal model such as described herein.

73. Monovalent construct, comprising or essentially consisting of oneamino acid sequence according to any of embodiments 1 to 40.

74. Monovalent construct according to embodiment 57, in which said aminoacid sequence of the invention is chosen from the group consisting ofdomain antibodies, amino acid sequences that are suitable for use as adomain antibody, single domain antibodies, amino acid sequences that aresuitable for use as a single domain antibody, “dAb”'s, amino acidsequences that are suitable for use as a dAb, or Nanobodies.

75. Monovalent construct, comprising or essentially consisting of oneNanobody according to any of embodiments 1 to 40.

76. Nucleic acid or nucleotide sequence, that encodes an amino acidsequence according to any of embodiments 1 to 40, a compound orconstruct according to any of embodiments 41 to 72, or a monovalentconstruct according to any of embodiments 73 to 75.

77. Nucleic acid or nucleotide sequence according to embodiment 76, thatis in the form of a genetic construct.

78. Host or host cell that expresses, or that under suitablecircumstances is capable of expressing, an amino acid sequence accordingto any of embodiments 1 to 40, a compound or construct according to anyof embodiments 41 to 72, or a monovalent construct according to any ofembodiments 73 to 75; and/or that comprises a nucleic acid or nucleotidesequence according to embodiment 76 or 77.

79. Method for producing an amino acid sequence according to any ofembodiments 1 to 40, a compound or construct according to any ofembodiments 41 to 72, or a monovalent construct according to any ofembodiments 73 to 75, said method at least comprising the steps of:

-   a) expressing, in a suitable host cell or host organism or in    another suitable expression system, a nucleic acid or nucleotide    sequence according to embodiment 76, or a genetic construct    according to embodiment 77; optionally followed by:-   b) isolating and/or purifying the amino acid sequence according to    any of embodiments 1 to 40, a compound or construct according to any    of embodiments 41 to 72, or a monovalent construct according to any    of embodiments 73 to 75.

80. Method for an amino acid sequence according to any of embodiments 1to 40, a compound or construct according to any of embodiments 41 to 72,or a monovalent construct according to any of embodiments 73 to 75, saidmethod at least comprising the steps of:

-   a) cultivating and/or maintaining a host or host cell according to    embodiment 78 under conditions that are such that said host or host    cell expresses and/or produces at least one amino acid sequence    according to any of embodiments 1 to 40, a compound or construct    according to any of embodiments 41 to 72, or a monovalent construct    according to any of embodiments 73 to 75; optionally followed by:-   b) isolating and/or purifying the amino acid sequence according to    any of embodiments 1 to 40, a compound or construct according to any    of embodiments 41 to 72, or a monovalent construct according to any    of embodiments 73 to 75.

81. Composition, comprising at least one an amino acid sequenceaccording to any of embodiments 1 to 40, a compound or constructaccording to any of embodiments 41 to 72, or a monovalent constructaccording to any of embodiments 73 to 75, or nucleic acid or nucleotidesequence according to embodiments 76 or 77.

82. Composition according to embodiment 81, which is a pharmaceuticalcomposition

83. Composition according to embodiment 82, which is a pharmaceuticalcomposition, that further comprises at least one pharmaceuticallyacceptable carrier, diluent or excipient and/or adjuvant, and thatoptionally comprises one or more further pharmaceutically activepolypeptides and/or compounds.

84. Method for the prevention and/or treatment of at least one diseaseor disorder related to excessive or insufficient angiogenesis, saidmethod comprising administering, to a subject in need thereof, apharmaceutically active amount of at least one amino acid sequenceaccording to any of embodiments 1 to 40, a compound or constructaccording to any of embodiments 41 to 72, or a monovalent constructaccording to any of embodiments 73 to 75, or composition according toany of embodiments 81 to 83.

85. Method for the prevention and/or treatment of at least one diseaseor disorder that is associated with a protein selected from the groupconsisting of Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2,Angptl3, Angptl4, Angptl5, and Angptl6, with its biological orpharmacological activity, and/or with the biological pathways orsignalling in which said protein is involved, said method comprisingadministering, to a subject in need thereof, a pharmaceutically activeamount of at least one amino acid sequence according to any ofembodiments 1 to 40, a compound or construct according to any ofembodiments 41 to 72, or a monovalent construct according to any ofembodiments 73 to 75, or composition according to any of embodiments 81to 83.

86. Method for the prevention and/or treatment of at least one diseaseor disorder related to cancer that can be prevented and/or treated byadministering, to a subject in need thereof, amino acid sequenceaccording to any of embodiments 1 to 40, a compound or constructaccording to any of embodiments 41 to 72, or a monovalent constructaccording to any of embodiments 73 to 75, or composition according toany of embodiments 81 to 83, said method comprising administering, to asubject in need thereof, a pharmaceutically active amount of at leastone amino acid sequence according to any of embodiments 1 to 40, acompound or construct according to any of embodiments 41 to 72, or amonovalent construct according to any of embodiments 73 to 75, orcomposition according to any of embodiments 81 to 83.

87. Use of an amino acid sequence according to any of embodiments 1 to40, a compound or construct according to any of embodiments 41 to 72, ora monovalent construct according to any of embodiments 73 to 75, in thepreparation of a pharmaceutical composition for prevention and/ortreatment of at least one disease or disorder related to excessive orinsufficient angiogenesis.

88. Use of an amino acid sequence according to any of embodiments 1 to40, a compound or construct according to any of embodiments 41 to 72, ora monovalent construct according to any of embodiments 73 to 75, for theprevention and/or treatment of at least one disease or disorder relatedto excessive or insufficient angiogenesis.

89. Use of an amino acid sequence according to any of embodiments 1 to40, a compound or construct according to any of embodiments 41 to 72, ora monovalent construct according to any of embodiments 73 to 75 for thetreatment of disease wherein excessive angiogenesis is the underlyingcause, and wherein the dosing regimen used is controlled in such a waythat the ratio of functional Ang1 to functional Ang2 in the serum of apatient is between 0.5 and 2, preferably between 0.6 and 1.67, morepreferably between 0.7 and 1.4, more preferably between 0.8 and 1.25,more preferably between 0.9 and 1.1.

90. Use according to embodiment 89, wherein the concentration offunctional Ang2 is considered to be the total concentration of Ang2 inserum minus the total concentration of the amino acid sequence directedagainst Ang2 in serum.

91. Method for the prevention and/or treatment of at least one diseaseor disorder related to cancer that can be prevented and/or treated byadministering, to a subject in need thereof, an amino acid sequenceaccording to any of embodiments 1 to 40, a compound or constructaccording to any of embodiments 41 to 72, or a monovalent constructaccording to any of embodiments 73 to 75, or composition according toany of embodiments 81 to 83, said method comprising administering, to asubject in need thereof, a pharmaceutically active amount of at leastone amino acid sequence according to any of embodiments 1 to 40, acompound or construct according to any of embodiments 41 to 72, or amonovalent construct according to any of embodiments 73 to 75, orcomposition according to any of embodiments 81 to 83; and wherein thedosing regimen used is controlled in such a way that the ratio offunctional Ang1 to functional Ang2 in the serum of a patient is between0.5 and 2, preferably between 0.6 and 1.67, more preferably between 0.7and 1.4, more preferably between 0.8 and 1.25, more preferably between0.9 and 1.1.

92. Method according to embodiment 91, wherein the concentration offunctional Ang2 is considered to be the total concentration of Ang2 inserum minus the total concentration of the amino acid sequence directedagainst Ang2 in serum.

Even More Preferred Aspects:

1. Amino acid sequence comprising at least one single variable domainthat is directed against a protein selected from the group consisting ofTie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4,Angptl5, and Angptl6.

2. Amino acid sequence according to any previous aspects, which is inessentially isolated form.

3. Amino acid sequence according to any previous aspects, wherein thevariable domain is directed against a protein selected from the groupconsisting of the human Tie1, human Tie2, human Ang1, human Ang2, humanAng3, human Ang4, human Angptl1, human Angptl2, human Angptl3, humanAngptl4, human Angptl5, and human Angptl6, preferably human Ang1, humanAng2, human Ang4, human Angptl4 and human Tie2.

4. Amino acid sequence according to any previous aspects, wherein thesingle variable domain has an antagonistic effect to at least one memberof the group of proteins consisting of Tie1 and Tie2.

5. Amino acid sequence according to any previous aspects, wherein thesingle variable domain has an antagonistic effect to human Tie2.

6. Amino acid sequence according to any previous aspects, wherein thesingle variable domain has an antagonistic effect to human Tie2 and doesnot block interaction between human Ang2 and human Tie2.

7. Amino acid sequence according to any previous aspects, wherein thesingle variable domain has the CDRs of SEQ ID NO 461.

8. Amino acid sequence according to any previous aspects, wherein thesingle variable domain has 80%, preferably 90%, more preferably 95%sequence identity with SEQ ID NO: 461.

9. Polypeptide comprising at least 2 identical or different amino acidsequence of any of the aspects 1 to 8.

10. Single variable domain with a CDR combination of any of the singlevariable domain in any of aspects 1 to 8, e.g. of SEQ ID NO: 461.

11. Single variable domain with 80%, preferably 90%, more preferably 95%sequence identity with any of the single variable domains in any ofaspects 1 to 8, e.g. SEQ ID NO: 461.

12. Pharmaceutical composition comprising an amino acid sequence, apolypeptide, or a single variable domain according to any previousaspects and at least one pharmaceutically acceptable carrier, diluent orexcipient and/or adjuvant.

13. Method for the prevention and/or treatment of at least one diseaseor disorder related to excessive or insufficient angiogenesis, saidmethod comprising administering, to a subject in need thereof, apharmaceutically active amount of at least one amino acid sequence, apolypeptide, or a single variable domain according to any of aspects 1to 11.

14. Use of an amino acid sequence, a polypeptide, or a single variabledomain according to any of aspects 1 to 11 for prevention and/ortreatment of at least one disease or disorder related to excessive orinsufficient angiogenesis.

15. Method for producing an amino acid sequence, a polypeptide, or asingle variable domain according to any of aspects 1 to 11, said methodat least comprising the steps of:

-   -   i. cultivating and/or maintaining a suitable host or host cell        under conditions that are such that said host or host cell        expresses and/or produces at least one amino acid sequence, a        polypeptide, or a single variable domain according to any of        aspects 1 to 11; optionally followed by:    -   ii. isolating and/or purifying the amino acid sequence, a        polypeptide, or a single variable domain according to any of        aspects 1 to 11.

1. Amino acid sequence comprising at least one single variable domainthat is directed against a protein selected from the group consisting ofTie2, Ang2, Tie1, Ang1, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4,Angptl5, and Angptl6.
 2. Amino acid sequence according to claim 1, whichis in essentially isolated form.
 3. Amino acid sequence according toclaim 1, wherein the variable domain is directed against a proteinselected from the group consisting of the human Tie2, human Ang2, humanTie1, human Ang1, human Ang3, human Ang4, human Angptl1, human Angptl2,human Angptl3, human Angptl4, human Angptl5, and human Angptl6,preferably human Ang1, human Ang2, human Ang4, human Angptl4 and humanTie2.
 4. Amino acid sequence according to claim 1, wherein the singlevariable domain has an antagonistic effect to at least one member of thegroup of proteins consisting of Tie2 and Tie1.
 5. Amino acid sequenceaccording to claim 2, wherein the single variable domain has anantagonistic effect to human Tie2.
 6. Amino acid sequence according toclaim 2, wherein the single variable domain has an antagonistic effectto human Tie2 and does not block interaction between human Ang2 andhuman Tie2.
 7. Amino acid sequence according to claim 1, wherein thesingle variable domain has the CDRs of SEQ ID NO;
 461. 8. Amino acidsequence according to claim 1, wherein the single variable domain has80%, preferably 90%, more preferably 95% sequence identity with SEQ IDNO:
 461. 9. Polypeptide comprising at least 2 identical or differentamino acid sequences of claim
 1. 10. Single variable domain with a CDRcombination of any of the single variable domain in claim 1, e.g. of SEQID NO:
 461. 11. Single variable domain with 80%, preferably 90%, morepreferably 95% sequence identity with any of the single variable domainsin claim 1, e.g. SEQ ID NO:
 461. 12. Pharmaceutical compositioncomprising an amino acid sequence according to claim 1 and at least onepharmaceutically acceptable carrier, diluent or excipient and/oradjuvant.
 13. Method for the prevention and/or treatment of at least onedisease or disorder related to excessive or insufficient angiogenesis,said method comprising administering, to a subject in need thereof, apharmaceutically active amount of at least one amino acid sequenceaccording to claim
 1. 14. Method for the prevention and/or treatment ofat least one disease or disorder related to excessive or insufficientangiogenesis, said method comprising administering, to a subject in needthereof, a pharmaceutically active amount of at least one a polypeptideaccording to claim
 9. 15. Method for producing an amino acid sequence, apolypeptide, or a single variable domain, said method at leastcomprising the steps of: i. cultivating and/or maintaining a suitablehost or host cell under conditions that are such that said host or hostcell expresses and/or produces at least one amino acid sequenceaccording to claim 1; optionally followed by: ii. isolating and/orpurifying the amino acid sequence according to claim 1.